Methods for identification of sites for igg conjugation

ABSTRACT

The present disclosure relates to immunoglobulins and immunoglobulin conjugates with reduced oligomerization and efficient labeling and compositions, methods of generating such immunoglobulins and immunoglobulin conjugates and methods of using such immunoglobulin conjugates particularly in the treatment and prevention of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/375,466, claiming an international filing date of Jun. 4, 2010; whichis a U.S. National Phase of International Patent Application No.PCT/US2010/037517, filed Jun. 4, 2010; which claims the benefit of U.S.Provisional Patent Application No. 61/184,084, filed Jun. 4, 2009; allof which are hereby incorporated by reference in the present disclosurein their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 619672000210SEQLIST.txt,date recorded: Jul. 30, 2014, size: 15 KB).

FIELD OF THE INVENTION

The present disclosure relates to improved immunoglobulins andimmunoglobulin conjugates.

BACKGROUND

Monoclonal antibodies are of great laboratory and therapeutic use.Antibody derivatives with engineered site-specific fluorescence orbinding properties have been developed and used for many years. Morerecently, antibodies have been also developed as therapeutic agents,currently presenting the fastest growing class of pharmaceuticals [1].Antibodies are multidomain proteins of two light and two heavy chainsheld together by disulfide bonds. The variable regions specify bindingto a particular antigen, and part of the constant regions is responsiblefor effector functions via binding to Fc receptors on the surface ofimmune cells. Because of their potential in the cure of variousdiseases, antibodies currently constitute the most rapidly growing classof human therapeutics (Carter. Nature Reviews Immunology. 2006, 6(5),343). Since 2001, their market has been growing at an average yearlygrowth rate of 35%, the highest rate among all categories of biotechdrugs (S. Aggarwal. Nature. BioTech. 2007, 25 (10) 1097).

Engineering of antibody conjugates has further increased the versatilityof antibody applications. In many laboratory techniques, enzymes orfluorescent probes are conjugated to antibodies to carry out an assayfunction, for example quantitation of antigen abundance. In cases oftargeted therapy, toxic small molecules are attached to antibodies thatspecifically bind biomarkers on diseased cells [2-4]. Various approachesto antibody conjugation have been pursued, for example attachment tosurface lysines [5], to Fc carbohydrates [6], or to partially reducedinterchain disulfides [7].

Antibody conjugation to engineered surface cysteine remains a veryattractive option because most antibodies do not have cysteines otherthan the ones consumed in intra- and inter-chain disulfide bonds. Smallmolecules can be attached at the specific site of cysteine substitutionvia a thiol reactive chemistry such as maleimides [8-14]. Engineering inthe C_(H)1 and C_(H)3 domains has been favored to avoid interferencewith antigen binding of the variable regions and effector function ofC_(H)2. Different criteria for successful antibody conjugation viaengineered cysteines have been considered. For example, the antibodydomain in which to carry out mutation, the exposure of the mutated site,the amino acid to be substituted are several of the variables to takeinto account. A high throughput screening approach to identifying sitessuitable for cysteine engineering and conjugation has been developed[15]. Two of the most common problems associated with antibody cysteinevariants are oligomerization and poor labeling. Yet, there is nouniversal tool for predicting whether an antibody cysteine variant willbe stable and efficiently conjugated. Furthermore, cysteine variantscurrently exist only for the C_(L), C_(H)1 and C_(H)3 domains [8, 9, 11,12, 15].

Thus, there is a need for additional immunoglobulin cysteine variantsthat can be used in the generation of stable immunoglobulin conjugates.

SUMMARY

Described herein are improved immunoglobulins and immunoglobulinconjugates which exhibit reduced cross-linking that meet this need.

Thus one aspect includes an immunoglobulin conjugate comprising animmunoglobulin having at least one mutation at a residue selected fromthe group consisting of 7(V_(H)), 20(V_(L)), 22(V_(L)), 25(V_(H)),125(C_(H1)), 248 (C_(H2)), 254(C_(H2)), 286(C_(H2)), 298(C_(H2)), and326(C_(H2)), wherein the at least one mutation is a substitution with acysteine residue, and an atom or molecule, wherein the atom or moleculeis conjugated to the cysteine residue. In certain embodiments, the atleast one mutation is at a residue selected from the group consisting of7(V_(H)), 20(V_(L)), 22(V_(L)) and 125(C_(H1)). In certain embodiments,the at least one mutation is at a residue selected from the groupconsisting of 248(C_(H2)) and 326(C_(H2)). In certain embodiments, theat least one mutation is at a residue selected from the group consistingof 25(V_(H)) and 286(C_(H2)). In certain embodiments, the at least onemutation is at residue selected from the group consisting of 254(C_(H2))and 298(V_(H)). In certain embodiments that may be combined with thepreceding embodiments, the immunoglobulin is selected from the groupcomprising IgG1, IgG2, IgG3, and IgG4. In certain embodiments that maybe combined with the preceding embodiments, the immunoglobulin comprisesan IgG1. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(H1)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(H2)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(H3)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(L)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human V_(H)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human V_(L)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate further comprises a linkermolecule having at least two reactive sites, wherein a first reactivesite is bound to the cysteine residue of the immunoglobulin and a secondreactive site is bound to the atom or molecule. In certain embodimentsthat may be combined with the preceding embodiments having a linkermolecule, the linker molecule is selected from the group consisting of ahydrazone, a disulfide, a peptide, a chelating agent, and a maleimide.In certain embodiments that may be combined with the precedingembodiments, the atom or molecule is selected from the group consistingof a radionuclide, a chemotherapeutic agent, a microbial toxin, a planttoxin, a polymer, a carbohydrate, a cytokine, a fluorescent label, aluminescent label, an enzyme-substrate label, an enzyme, a peptide, apeptidomimetic, a nucleotide, an siRNA, a microRNA, an RNA mimetic, andan aptamer. In certain embodiments that may be combined with thepreceding embodiments, the atom or molecule is selected from the groupconsisting of ⁹⁰Y, ¹³¹I ⁶⁷Cu, ¹⁷⁷Lu, ²¹³Bi, ²¹¹At, a calicheamicin, aduocarmycin, a maytanisoid, an auristatin, an anthracyclin, Pseudomonasexotoxin A, Diptheria toxin, ricin, polyethylene glycol, hydroxyethylstarch, and a mannosyl residue. In certain embodiments that may becombined with the preceding embodiments, the atom or molecule reducesthe immunogenicity of the unmutated immunoglobulin. In certainembodiments that may be combined with the preceding embodiments, theatom or molecule increases the immunogenicity of the unmutatedimmunoglobulin. In certain embodiments that may be combined with thepreceding embodiments, the immunoglobulin conjugate further comprises anantigen binding activity and the activity is at least eighty percent, atleast ninety percent, at least one hundred percent, at least one hundredten percent, at least one hundred twenty percent, or at least onehundred thirty percent of the antigen binding activity of the unmutatedimmunoglobulin.

Another aspect includes a modified or isolated immunoglobulin comprisingat least one mutation at a residue selected from the group consisting of7(V_(H)), 20(V_(L)), 22(V_(L)), 25(V_(H)), 125(C_(H1)), 248(C_(H2)),254(C_(H2)), 286(C_(H2)), and 326(C_(H2)), wherein the at least onemutation is a substitution with a cysteine residue. In certainembodiments, the at least one mutation is at a residue selected from thegroup consisting of 7(V_(H)), 20(V_(L)), 22(V_(L)) and 125(C_(H1)). Incertain embodiments, the at least one mutation is at a residue selectedfrom the group consisting of 248(C_(H2)) and 326(C_(H2)). In certainembodiments, the at least one mutation is at a residue selected from thegroup consisting of 25(V_(H)) and 286(C_(H2)). In certain embodiments,the at least one mutation is at residue 254(C_(H2)). In certainembodiments that may be combined with the preceding embodiments, theimmunoglobulin is selected from the group comprising IgG1, IgG2, IgG3,and IgG4. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin comprises an IgG1. In certainembodiments that may be combined with the preceding embodiments, themodified or isolated immunoglobulin comprises a human C_(H1) domain. Incertain embodiments that may be combined with the preceding embodiments,the modified or isolated immunoglobulin comprises a human C_(H2) domain.In certain embodiments that may be combined with the precedingembodiments, the modified or isolated immunoglobulin comprises a humanC_(H3) domain. In certain embodiments that may be combined with thepreceding embodiments, the modified or isolated immunoglobulin comprisesa human C_(L) domain. In certain embodiments that may be combined withthe preceding embodiments, the modified or isolated immunoglobulincomprises a human V_(H) domain. In certain embodiments that may becombined with the preceding embodiments, the modified or isolatedimmunoglobulin comprises a human V_(L) domain. In certain embodimentsthat may be combined with the preceding embodiments, the immunoglobulinfurther comprises an antigen binding activity and the activity is atleast eighty percent, at least ninety percent, at least one hundredpercent, at least one hundred ten percent, at least one hundred twentypercent, or at least one hundred thirty percent of the antigen bindingactivity of the unmutated immunoglobulin.

Another aspect includes isolated or recombinant polynucleotides thatencode the immunoglobulins of the preceding modified immunoglobulinaspect and any and all combinations of the preceding embodiments. Incertain embodiments, the polynucleotide is in a vector. In certainembodiments, the vector is an expression vector. In certain embodimentsthat may be combined with the preceding embodiments, an induciblepromoter is operably linked to the polynucleotide. Another aspectincludes host cells with the vector of either of the precedingembodiments. In certain embodiments, the host cells are capable ofexpressing the immunoglobulin encoded by the polynucleotide.

Another aspect includes methods of producing an immunoglobulincomprising providing a culture medium comprising the host cell of thepreceding aspect and placing the culture medium in conditions underwhich the immunoglobulin is expressed. In certain embodiments, themethods include an additional step of isolating the immunoglobulinexpressed.

Another aspect includes methods of producing an immunoglobulin conjugatecomprising providing the immunoglobulin of the preceding modifiedimmunoglobulin aspect and any and all combinations of the precedingembodiments, reducing the one or more substituted cysteine residues witha reducing agent to form reduced cysteine residues, and incubating theimmunoglobulin with an atom or molecule, wherein the atom or molecule isreactive with the reduced cysteine residues, to form an immunoglobulinconjugate.

Another aspect includes methods for reducing the cross-linking betweensurface-exposed cysteines of an immunoglobulin in a highly concentratedpharmaceutical formulation of immunoglobulin conjugates comprisingproviding an immunoglobulin, substituting a residue selected from thegroup consisting of 7(V_(H)), 20(V_(L)), 22(V_(L)), and 125(C_(H1)) witha cysteine residue, reducing the one or more substituted cysteineresidues with a reducing agent to form reduced cysteine residues,incubating the immunoglobulin with an atom or molecule, wherein themolecule is reactive with the reduced cysteine residues, to form animmunoglobulin conjugate, and generating a highly concentrated, liquidformulation of the immunoglobulin conjugate wherein the immunoglobulinconjugate concentration is at least 20 mg/ml, at least 30 mg/ml, atleast 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100mg/ml, at least 125 mg/ml, or at least 150 mg/ml. In certainembodiments, the immunoglobulin is selected from the group comprisingIgG1, IgG2, IgG3, and IgG4. In certain embodiments, the immunoglobulincomprises an IgG1. In certain embodiments that may be combined with thepreceding embodiments, the immunoglobulin comprises a human C_(H1)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin comprises a human C_(H2) domain. Incertain embodiments that may be combined with the preceding embodiments,the immunoglobulin comprises a human C_(H3) domain. In certainembodiments that may be combined with the preceding embodiments, theimmunoglobulin comprises a human C_(L) domain. In certain embodimentsthat may be combined with the preceding embodiments, the immunoglobulincomprises a human V_(H) domain. In certain embodiments that may becombined with the preceding embodiments, the immunoglobulin comprises ahuman V_(L) domain. In certain embodiments that may be combined with thepreceding embodiments, the immunoglobulin conjugate comprises an antigenbinding activity and the activity is at least eighty percent, at leastninety percent, at least one hundred percent, at least one hundred tenpercent, at least one hundred twenty percent, or at least one hundredthirty percent of the antigen binding activity of the unmutatedimmunoglobulin.

Another aspect includes uses of the preceding immunoglobulin conjugateaspect and any and all combinations of the preceding embodiments in thepreparation of a medicament comprising a highly concentrated liquidformulation wherein the immunoglobulin conjugate is at least 20 mg/ml,at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml. Incertain embodiments, the use of the medicament is for the treatment ofautoimmune diseases, immunological diseases, infectious diseases,inflammatory diseases, neurological diseases, and oncological andneoplastic diseases including cancer. In certain embodiments, the use ofthe medicament is for the treatment of congestive heart failure (CHF),vasculitis, rosacea, acne, eczema, myocarditis and other conditions ofthe myocardium, systemic lupus erythematosus, diabetes, spondylopathies,synovial fibroblasts, and bone marrow stroma; bone loss; Paget'sdisease, osteoclastoma; breast cancer; disuse osteopenia; malnutrition,periodontal disease, Gaucher's disease, Langerhans' cell histiocytosis,spinal cord injury, acute septic arthritis, osteomalacia, Cushing'ssyndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia,periodontal reconstruction, and bone fractures; sarcoidosis; osteolyticbone cancers, breast cancer, lung cancer, kidney cancer and rectalcancer; bone metastasis, bone pain management, and humoral malignanthypercalcemia, ankylosing spondylitisa and other spondyloarthropathies;transplantation rejection, viral infections, hematologic neoplasias andneoplastic-like conditions for example, Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticlymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplamacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, Langerhans cellhistocytosis, myeloid neoplasias such as acute myelogenous leukemias,including AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia,tumors of the central nervous system, e.g., brain tumors (glioma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma), solid tumors (nasopharyngeal cancer, basal cellcarcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer,primary liver cancer or endometrial cancer, and tumors of the vascularsystem (angiosarcoma and hemangiopericytoma), osteoporosis, hepatitis,HIV, AIDS, spondylarthritis, rheumatoid arthritis, inflammatory boweldiseases (IBD), sepsis and septic shock, Crohn's Disease, psoriasis,schleraderma, graft versus host disease (GVHD), allogenic islet graftrejection, hematologic malignancies, such as multiple myeloma (MM),myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML),inflammation associated with tumors, peripheral nerve injury ordemyelinating diseases. In certain embodiments, the use of themedicament is for the treatment of plaque psoriasis, ulcerative colitis,non-Hodgkin's lymphoma, breast cancer, colorectal cancer, juvenileidiopathic arthritis, macular degeneration, respiratory syncytial virus,Crohn's disease, rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, osteoporosis, treatment-induced bone loss, bone metastases,multiple myeloma, Alzheimer's disease, glaucoma, and multiple sclerosis.In certain embodiments that may be combined with the precedingembodiments, the medicament further comprises a pharmaceuticallyacceptable excipient. In certain embodiments that may be combined withthe preceding embodiments, the formulation comprises at least eightypercent, at least eighty-five percent, at least ninety percent, at leastninety-five percent, at least ninety-six percent, at least ninety-sevenpercent, at least ninety-eight percent, or at least ninety-nine percentof the immunoglobulin conjugate is non-oligomerized monomer. In certainembodiments, the percentage of monomers is measured by non-reducingSDS-PAGE analysis.

Another aspect includes uses of the preceding immunoglobulin conjugateaspect and any and all combinations of the preceding embodiments as anon-oligomerizing pharmaceutical active ingredient.

Another aspect includes uses of the preceding immunoglobulin conjugateaspect and any and all combinations of the preceding embodiments as adiagnostic tool.

Another aspect includes uses of the preceding immunoglobulin conjugateaspect and any and all combinations of the preceding embodiments as astandard for high molecular weight proteins. Another aspect includesuses of an immunoglobulin conjugate as a standard for high molecularweight proteins, wherein the immunoglobulin conjugate comprises animmunoglobulin having at least one mutation at residue 440(C_(H3)),wherein the at least one mutation is a substitution with a cysteineresidue, and an atom or molecule, wherein the atom or molecule isconjugated to the cysteine residue.

Another aspect includes pharmaceutical compositions that include animmunoglobulin conjugate of the preceding immunoglobulin conjugateaspect and any and all combinations of the preceding embodiments and apharmaceutically acceptable excipient. In certain embodiments, theimmunoglobulin conjugate is at a concentration of at least 10 mg/ml, atleast 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml,at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least150 mg/ml. In certain embodiments that may be combined with thepreceding embodiments, at least eighty percent, at least eighty-fivepercent, at least ninety percent, at least ninety-five percent, at leastninety-six percent, at least ninety-seven percent, at least ninety-eightpercent, or at least ninety-nine percent of the immunoglobulin conjugateis non-oligomerized monomer.

Another aspect includes methods for selecting a residue of animmunoglobulin for mutation to cysteine comprising calculating theSpatial-Aggregation-Propensity of a first amino acid residue on thesurface of the immunoglobulin, calculating theSpatial-Aggregation-Propensities of a plurality of residues of theimmunoglobulin within immediate proximity of the first residue, andselecting the first amino acid residue for mutation to cysteine if theSpatial-Aggregation-Propensity of the first amino acid residue is equalto or in between the values of 0 and −0.11 and if the plurality ofresidues have Spatial-Aggregation-Propensities of less than 0. Incertain embodiments, the plurality of residues is within 15 Å of thefirst residue. In certain embodiments, the plurality of residues iswithin 10 Å of the first residue. In certain embodiments, the pluralityof residues is within 7.5 Å of the first residue. In certainembodiments, the plurality of residues is within 5 Å of the firstresidue. In certain embodiments that may be combined with the precedingembodiments, calculating the Spatial-Aggregation-Propensity of a residuecomprises calculating the Spatial-Aggregation-Propensity for a sphericalregion with a radius centered on an atom in the residue. In certainembodiments, the radius of the spherical region is at least 5 Å.

Another aspect includes modified or isolated immunoglobulins comprisingat least one mutation of a surface-exposed residue to cysteine, whereinthe Spatial-Aggregation-Propensity of the residue is equal to or inbetween the values of 0 and −0.11 and wherein theSpatial-Aggregation-Propensities of a plurality of residues of theimmunoglobulin within immediate proximity of the first residue haveSpatial-Aggregation-Propensities of less than 0. In certain embodiments,the plurality of residues is within 15 Å of the first residue. Incertain embodiments, the plurality of residues is within 10 Å of thefirst residue. In certain embodiments, the plurality of residues iswithin 7.5 Å of the first residue. In certain embodiments, the pluralityof residues is within 5 Å of the first residue. In certain embodimentsthat may be combined with the preceding embodiments, theSpatial-Aggregation-Propensity is calculated for a spherical region witha radius centered on an atom in the residue. In certain embodiments, theradius is at least 5 Å.

Another aspect includes methods of selecting a residue of animmunoglobulin for mutation to cysteine comprising choosing a pluralityof amino acid residues of the immunoglobulin, wherein the plurality ofresidues are exposed on the surface of the immunoglobulin, mutating oneresidue of the plurality of residues to a cysteine residue, conjugatingthe cysteine residue to an atom or molecule to form an immunoglobulinconjugate, testing the immunoglobulin conjugate for cross-linkingpropensity and assigning the immunoglobulin conjugate a cross-linkingpropensity value, and selecting the residue for mutation to cysteine ifthe cross-linking propensity value is I or II. In certain embodiments,the method further comprises assigning the immunoglobulin conjugate across-linking propensity value of II if less than 5% of theimmunoglobulin conjugate forms dimers and none of the immunoglobulinconjugate forms trimers wherein dimer and trimer formation is measuredby comparative non-reducing and reducing SDS-PAGE. In certainembodiments, the method further comprises assigning the immunoglobulinconjugate a cross-linking propensity value of I if less than 1% of theimmunoglobulin conjugate forms dimers wherein dimer formation ismeasured by comparative non-reducing and reducing SDS-PAGE.

Additional aspects and embodiments of the invention may be foundthroughout the specification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present disclosure relates to improved immunoglobulins andimmunoglobulin conjugates which exhibit reduced cross-linking. Incertain embodiments, the immunoglobulins of the disclosure are modifiedat specific residues by substitution with cysteine. The disclosureprovides modified immunoglobulins and immunoglobulin conjugates, methodsof making such immunoglobulins and immunoglobulin conjugates,multivalent or multispecific molecules comprising such immunoglobulinsand pharmaceutical compositions containing the immunoglobulins,immunoglobulin conjugates or bispecific molecules of the disclosure.

DEFINITIONS

The term “antibody” or “immunoglobulin” as referred to herein includeswhole antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chains thereof. A naturallyoccurring “antibody” is a glycoprotein comprising at least two heavy (H)chains and two light (L) chains inter-connected by disulfide bonds. Eachheavy chain is comprised of a heavy chain variable region (abbreviatedherein as V_(H)) and a heavy chain constant region. The heavy chainconstant region is comprised of three domains, C_(H1), C_(H2) andC_(H3). Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, C_(L). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The terms “antibody conjugate” or “immunoglobulin conjugate” as referredto herein include any immunoglobulin, antigen binding fragment, orsingle chains thereof chemically or biologically linked to an atom ormolecule. Atoms or molecules may include, for example, a cytotoxin,radioactive agent, anti-tumor drug, or therapeutic agent. The antibodyconjugate retains the immunoreactivity of the immunoglobulin or antigenbinding fragment, i.e., the immunoglobulin or antigen binding fragmentof the antibody conjugate has at least seventy percent, at leastseventy-five percent, at least eighty percent, at least eighty-fivepercent, at least ninety percent, at least ninety-five percent, at leastat least one hundred percent, at least one hundred ten percent, at leastone hundred twenty percent, or at least one hundred thirty percent ofthe antigen binding activity of the immunoglobulin prior to conjugationwith the atom or molecule.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen and at least a portion of the constant region of the heavy orlight chain. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; a F(ab)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and C_(H1) domains; and a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding region” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated” antibody or immunoglobulin, as used herein, refers to anantibody or immunoglobulin that is substantially free of othercomponents in which such antibodies or immunoglobulin are naturallyfound. Moreover, an isolated antibody or immunoglobulin may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition typicallydisplays a single binding specificity and affinity for a particularepitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences or antibody containing consensusframework sequences derived from human framework sequences analysis asdescribed in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The human antibodies of the disclosure may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human domain”, as used herein, is intended to includeimmunoglobulin constant region domains derived from sequences of humanorigin, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis as described in Knappik,et al. (2000. J Mol Biol 296, 57-86).

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. For example, a mouse antibody can bemodified by replacing its constant region with the constant region froma human immunoglobulin comprising a modification as disclosed herein.Due to the replacement with a human constant region, the chimericantibody can retain its specificity while having reduced antigenicity inhuman and reduced aggregation overall as compared to the original mouseantibody or a chimeric antibody without the modification as disclosedherein.

A “humanized” antibody is an antibody that retains the reactivity of anon-human antibody while being less immunogenic in humans. This can beachieved, for instance, by retaining the non-human CDR regions andreplacing the remaining parts of the antibody with their humancounterparts (i.e., the constant region as well as the frameworkportions of the variable region). See, e.g., Morrison et al., Proc.Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv.Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536,1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun,31:169-217, 1994. Other examples of human engineering technologyinclude, but are not limited to Xoma technology disclosed in U.S. Pat.No. 5,766,886.

The term “linker”, “linker Unit”, or “link” as used herein refers to achemical moiety comprising a covalent bond or a chain of atoms thatcovalently attaches an antibody to a drug moiety or other molecule.Linkers include a divalent radical such as an alkyldiyl, an arylene, aheteroarylene, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g. polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

The term “label” as used herein refers to any moiety which can becovalently attached to an antibody and that functions to: (i) provide adetectable signal; (ii) interact with a second label to modify thedetectable signal provided by the first or second label, e.g. FRET(fluorescence resonance energy transfer); (iii) stabilize interactionsor increase affinity of binding, with antigen or ligand; (iv) affectmobility, e.g. electrophoretic mobility, or cell-permeability, bycharge, hydrophobicity, shape, or other physical parameters, or (v)provide a capture moiety, to modulate ligand affinity, antibody/antigenbinding, or ionic complexation.

The term “Humaneering” as used herein refers to a method for convertingnon-human antibodies into engineered human antibodies (See e.g.,KaloBios' Humaneering™ technology).

As used herein, “isotype” refers to any antibody class (e.g., IgM, IgE,IgG such as IgG1 or IgG2) that is provided by the heavy chain constantregion genes that have the aggregation prone motifs disclosed herein(and therefore are amenable to the modifications disclosed herein thatreduce aggregation).

As used herein, the term “affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity. Themodifications disclosed herein preferably do not reduce the affinity ofthe immunoglobulin or antibodies disclosed herein or the affinity isreduced less than thirty percent, less than twenty percent, less thanten percent, or less than five percent. As used herein, when determiningwhether the modifications disclosed herein reduce affinity thecomparison is made between the immunoglobulin or antibody with themodification and the same immunoglobulin lacking the modification butincluding any unrelated mutations.

As used herein, the term “subject” includes any human or nonhumananimal.

The term “nonhuman animal” includes all vertebrates, e g, mammals andnon-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows,chickens, amphibians, reptiles, etc.

The term “chemotherapeutic agent” as used herein refers to a chemicalcompound useful in the treatment of cancer. Examples of chemotherapeuticagents include Erlotinib (TARCEVA™, Genentech/OSI Pharm.), Bortezomib(VELCADE™, Millenium Pharm.), Fulvestrant (FASLODEX™, Astrazeneca),Sutent (SU11248, Pfizer), Letrozole (FEMARA™, Novartis), Imatinibmesylate (GLEEVEC™, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin™, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE™, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline),Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), andGefitinib (IRESSA™, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen),alkylating agents such as thiotepa and CYTOXAN™ cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall (Angew ChemIntl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, anthramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL™paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR™ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX™tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON toremifene; (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE™ megestrol acetate, AROMASIN™exemestane, formestanie, fadrozole, RIVISOR™ vorozole, FEMARA™letrozole, and ARIMIDEX™ anastrozole; (iii) anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv)aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinaseinhibitors; (vii) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;(viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME™ribozyme) and a HER2 expression inhibitor; (ix) vaccines such as genetherapy vaccines, for example, ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine,and VAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™ topoisomerase 1inhibitor; ABARELIX™ rmRH; (x) anti-angiogenic agents such asbevacizumab (AVASTIN™, Genentech); and (xi) pharmaceutically acceptablesalts, acids or derivatives of any of the above.

As used herein, the term “cytokine” is a generic term for proteinsreleased by one cell population which act on another cell asintercellular mediators. Examples of such cytokines are lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are growth hormone such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;fibroblast growth factor; prolactin; placental lactogen; tumor necrosisfactor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumornecrosis factor such as TNF-α or TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO)or a human cell. The optimized nucleotide sequence is engineered toretain completely or as much as possible the amino acid sequenceoriginally encoded by the starting nucleotide sequence, which is alsoknown as the “parental” sequence. Optimized expression of thesesequences in other eukaryotic cells is also envisioned herein. The aminoacid sequences encoded by optimized nucleotide sequences are alsoreferred to as optimized.

As used herein, the term “antigen binding activity” refers to thespecificity of binding of an immunoglobulin or immunoglobulin conjugateto its target antigen. For example, antigen binding activity may bemeasured by cell-based bioassays (e.g. reporter gene assays), ELISA,surface plasmon resonance (Biacore), or any other techniques known toone skilled in the art.

As used herein, the term “cross-linking propensity” (CLP) refers to thepropensity of a modified immunoglobulin or immunoglobulin conjugatecontaining a mutation that is a substitution with cysteine to cross-linkbetween different immunoglobulins at the substituted cysteine residue.For example, CLP can be determined by the level of oligomerization asmeasured by non-reducing SDS-PAGE, size-exclusion chromatography, staticor dynamic laser light scattering with size-exclusion chromatography, orany other techniques known to one skilled in the art. Class I comprisesvariants that are monomeric and remain stable after labeling. Variantsof class II contain a small percent of dimers before and after labeling.Class III variants have a more pronounced propensity to oligomerizeincluding formation of some trimers. Class IV variants have even higherpropensity to oligomerize as evidenced by the presence of aggregateslarger than trimer, especially after labeling. Class V includes variantsof high oligomerization propensity similarly to variant of Class IV withadditional structural abnormalities such as fragmentation or colorationof purified concentrated sample.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of thedisclosure. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. When comparing two sequences foridentity, it is not necessary that the sequences be contiguous, but anygap would carry with it a penalty that would reduce the overall percentidentity. For blastn, the default parameters are Gap opening penalty=5and Gap extension penalty=2. For blastp, the default parameters are Gapopening penalty=11 and Gap extension penalty=1.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions including, but notlimited to from 20 to 600, usually about 50 to about 200, more usuallyabout 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, it refers tothe functional relationship of a transcriptional regulatory sequence toa transcribed sequence. For example, a promoter or enhancer sequence isoperably linked to a coding sequence if it stimulates or modulates thetranscription of the coding sequence in an appropriate host cell orother expression system. Generally, promoter transcriptional regulatorysequences that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the disclosure is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “target antigen” refers to the antigen against which the parentimmunoglobulin was raised or otherwise generated (e.g., by phagedisplay).

The term “unmutated immunoglobulin” refers to the immunoglobulin whichdoes not comprise the at least one mutation that is a substitution witha cysteine residue. As used herein, the unmutated immunoglobulin may bea hypothetical construct for the purposes of comparison of theoligomerization propensity or the conjugation efficiency of theimmunoglobulin with and without the mutation. By way of example, amurine antibody that includes humanizing mutations as well as mutationsto cysteine for the purpose of conjugation is not the unmutatedimmunoglobulin. The unmutated immunoglobulin would be the antibody withthe humanizing mutations, but without the mutations to cysteine. Where amutation is intended to serve more than one purpose including providingsites for conjugation, the unmutated immunoglobulin does not includesuch mutation.

The term “aggregation motif” refers to a set of residues groupedtogether based upon the following process. First, residues having an SAP(5 Å radius) of greater than 0.15 are identified. Then all residueswithin 5 Å of each residue having an SAP (5 Å radius) of greater than0.15 are identified. A motif is then the residue with an SAP (5 Åradius) of greater than 0.15 and all residues with an SAP (5 Å radius)of greater than 0.0 within 5 Å of the residue with an SAP (5 Å radius)of greater than 0.15. Any such motifs having at least one residue incommon are merged into a larger motif reiteratively until there are noremaining motifs which have a residue in common. These remaining motifsor sets of residues constitute aggregation motifs.

Where immunoglobulin residues are referred to by number herein, theresidue number refers to the Kabat number of the corresponding residuein the IgG1 molecule when the immunoglobulin sequence of interest isaligned to the human IgG1 immunoglobulin. By way of reference, the humanIgG1, IgG2, IgG3 and IgG4 constant domains are aligned:

C_(H1) domain

(IgG1 = SEQ ID NO: 1; IgG2 = SEQ ID NO: 2; IgG4 = SEQ ID NO: 3; IgG3 =SEQ ID NO: 4) Hinge

C_(H2) domain

(IgG1 = SEQ ID NO: 9; IgG2 = SEQ ID NO: 10; IgG4 = SEQ ID NO: 11; IgG3 =SEQ ID NO: 12) C_(H3) domain

(IgG1 = SEQ ID NO: 13; IgG2 = SEQ ID NO: 14; IgG4 = SEQ ID NO: 15; IgG3= SEQ ID NO: 16) C_(L) domain

Alignments of the V_(H) and V_(L) domains can be found in Ewert,Honegger, and Plúchthun, Methods 34 (2004) 184-199.

Immunoglobulin Conjugates of the Invention

The invention herein relates to immunoglobulin conjugates includingimmunoglobulins having at least one mutation of a residue of the surfaceof the immunoglobulin wherein the mutation is a substitution with acysteine residue. The substituted cysteine residue is conjugated to anatom or molecule, which may be, by way of example, a cytotoxic agent(e.g. a toxin such as doxorubicin or pertussis toxin), a fluorophoresuch as a fluorescent dye like fluorescein or rhodamine, a chelatingagent for an imaging or radiotherapeutic metal, a peptidyl ornon-peptidyl label or detection tag, or a clearance-modifying agent suchas various isomers of polyethylene glycol, a peptide that binds to athird component, or another carbohydrate or lipophilic agent. In furtherembodiments, the molecule may be an enzyme, a peptide, a peptidomimetic,a nucleotide such as an RNA molecule, including siRNA, microRNA, and RNAmimetics, or aptamers.

Labeled Immunoglobulin Conjugates

In certain embodiments, modified immunoglobulins of the invention may beconjugated with any label moiety which can be covalently attached to theimmunoglobulin through a reactive cysteine thiol group (Singh et al(2002) Anal. Biochem. 304:147-15; Harlow E. and Lane, D. (1999) UsingAntibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press,Cold Spring Harbor, N.Y.; Lundblad R. L. (1991) Chemical Reagents forProtein Modification, 2nd ed. CRC Press, Boca Raton, Fla.). The attachedlabel may function, for example, to: (i) provide a detectable signal;(ii) interact with a second label to modify the detectable signalprovided by the first or second label, e.g. to give FRET (fluorescenceresonance energy transfer); (iii) stabilize interactions or increaseaffinity of binding, with antigen or ligand; (iv) affect mobility, e.g.electrophoretic mobility or cell-permeability, by charge,hydrophobicity, shape, or other physical parameters, or (v) provide acapture moiety, to modulate ligand affinity, antibody/antigen binding,or ionic complexation.

Labeled immunoglobulin conjugates may be useful in diagnostic assays,e.g., for detecting expression of an antigen of interest in specificcells, tissues, or serum. For diagnostic applications, theimmunoglobulin will typically be labeled with a detectable moiety.Numerous labels are available which can be generally grouped into thefollowing exemplary categories:

(a) Radioisotopes (radionuclides), such as ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S,⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu,²¹¹At, or ²¹³Bi. Radioisotope labeled immunoglobulins are useful inreceptor targeted imaging experiments. The immunoglobulin can be labeledwith ligand reagents that bind, chelate or otherwise complex aradioisotope metal where the reagent is reactive with the engineeredcysteine thiol of the immunoglobulin, using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991). Chelating ligandswhich may complex a metal ion include DOTA, DOTP, DOTMA, DTPA and TETA(Macrocyclics, Dallas, Tex.). Radionuclides can be targeted viacomplexation with the antibody-drug conjugates of the invention (Wu etal (2005) Nature Biotechnology 23(9):1137-1146).

Metal-chelate complexes suitable as immunoglobulin labels for imagingexperiments are disclosed: U.S. Pat. Nos. 5,342,606; 5,428,155;5,316,757; 5,480,990; 5,462,725; 5,428,139; 5,385,893; 5,739,294;5,750,660; 5,834,456; Hnatowich et al (1983) J. Immunol. Methods65:147-157; Meares et al (1984) Anal. Biochem. 142:68-78; Mirzadeh et al(1990) Bioconjugate Chem. 1:59-65; Meares et al (1990) J. Cancer 1990,Suppl. 10:21-26; Izard et al (1992) Bioconjugate Chem. 3:346-350; Nikulaet al (1995) Nucl. Med. Biol. 22:387-90; Camera et al (1993) Nucl. Med.Biol. 20:955-62; Kukis et al (1998) J. Nucl. Med. 39:2105-2110; Verel etal (2003) J. Nucl. Med. 44:1663-1670; Camera et al (1994) J. Nucl. Med.21:640-646; Ruegg et al (1990) Cancer Res. 50:4221-4226; Verel et al(2003) J. Nucl. Med. 44:1663-1670; Lee et al (2001) Cancer Res.61:4474-4482; Mitchell, et al (2003) J. Nucl. Med. 44:1105-1112;Kobayashi et al (1999) Bioconjugate Chem. 10:103-111; Miederer et al(2004) J. Nucl. Med. 45:129-137; DeNardo et al (1998) Clinical CancerResearch 4:2483-90; Blend et al (2003) Cancer Biotherapy &Radiopharmaceuticals 18:355-363; Nikula et al (1999) J. Nucl. Med.40:166-76; Kobayashi et al (1998) J. Nucl. Med. 39:829-36; Mardirossianet al (1993) Nucl. Med. Biol. 20:65-74; Roselli et al (1999) CancerBiotherapy & Radiopharmaceuticals, 14:209-20.

(b) Fluorescent labels such as rare earth chelates (europium chelates),fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types including TAMRA; dansyl; Lissamine;cyanines; phycoerythrins; Texas Red; and analogs thereof. Thefluorescent labels can be conjugated to immunoglobulins using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescent dyes and fluorescent label reagents include thosewhich are commercially available from Invitrogen/Molecular Probes(Eugene, Oreg.) and Pierce Biotechnology, Inc. (Rockford, Ill.).

c) Various enzyme-substrate labels are available or disclosed (U.S. Pat.No. 4,275,149). The enzyme generally catalyzes a chemical alteration ofa chromogenic substrate that can be measured using various techniques.For example, the enzyme may catalyze a color change in a substrate,which can be measured spectrophotometrically. Alternatively, the enzymemay alter the fluorescence or chemiluminescence of the substrate.Techniques for quantifying a change in fluorescence are described above.The chemiluminescent substrate becomes electronically excited by achemical reaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase(AP), β-galactosidase, glucoamylase, lysozyme, saccharide oxidases(e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al(1981) “Methods for the Preparation of Enzyme-Antibody Conjugates foruse in Enzyme Immunoassay”, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic Press, New York, 73:147-166.

Examples of enzyme-substrate combinations include, for example: (i)Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB)); (ii) alkaline phosphatase (AP) withpara-nitrophenyl phosphate as chromogenic substrate; and (iii)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase. Numerous other enzyme-substratecombinations are available to those skilled in the art. For a generalreview, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

A label may be indirectly conjugated with modified immunoglobulins ofthe invention. For example, the immunoglobulin can be conjugated withbiotin and any of the three broad categories of labels mentioned abovecan be conjugated with avidin or streptavidin, or vice versa. Biotinbinds selectively to streptavidin and thus, the label can be conjugatedwith the immunoglobulin in this indirect manner. Alternatively, toachieve indirect conjugation of the label with the immunoglobulinvariant, the immunoglobulin variant is conjugated with a small hapten(e.g., digoxin) and one of the different types of labels mentioned aboveis conjugated with an anti-hapten polypeptide variant (e.g.,anti-digoxin antibody). Thus, indirect conjugation of the label with theimmunoglobulin variant can be achieved (Hermanson, G. (1996) inBioconjugate Techniques Academic Press, San Diego).

The modified immunoglobulins and immunoglobulin conjugates of thepresent invention may be employed in any known assay method, such asELISA, competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: AManual of Techniques, pp. 147-158, CRC Press, Inc.).

A detection label may be useful for localizing, visualizing, andquantitating a binding or recognition event. The labeled immunoglobulinconjugates of the invention can detect cell-surface receptors. Anotheruse for detectably labeled immunoglobulin conjugates is a method ofbead-based immunocapture comprising conjugating a bead with afluorescent labeled antibody and detecting a fluorescence signal uponbinding of a ligand. Similar binding detection methodologies utilize thesurface plasmon resonance (SPR) effect to measure and detectantibody-antigen interactions.

Detection labels such as fluorescent dyes and chemiluminescent dyes(Briggs et al (1997) “Synthesis of Functionalised Fluorescent Dyes andTheir Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans.1:1051-1058) provide a detectable signal and are generally applicablefor labeling immunoglobulins, preferably with the following properties:(i) the labeled immunoglobulin should produce a very high signal withlow background so that small quantities of immunoglobulins can besensitively detected in both cell-free and cell-based assays; and (ii)the labeled antibody should be photostable so that the fluorescentsignal may be observed, monitored and recorded without significant photobleaching. For applications involving cell surface binding of labeledantibody to membranes or cell surfaces, especially live cells, thelabels preferably (iii) have good water-solubility to achieve effectiveconjugate concentration and detection sensitivity and (iv) are non-toxicto living cells so as not to disrupt the normal metabolic processes ofthe cells or cause premature cell death.

Direct quantification of cellular fluorescence intensity and enumerationof fluorescently labeled events, e.g. cell surface binding ofpeptide-dye conjugates may be conducted on an system (FMAT™ 8100 HTSSystem, Applied Biosystems, Foster City, Calif.) that automatesmix-and-read, non-radioactive assays with live cells or beads (Miraglia,“Homogeneous cell- and bead-based assays for high throughput screeningusing fluorometric microvolume assay technology”, (1999) J. ofBiomolecular Screening 4:193-204). Uses of labeled immunoglobulins alsoinclude cell surface receptor binding assays, immunocapture assays,fluorescence linked immunosorbent assays (FLISA), caspase-cleavage(Zheng, “Caspase-3 controls both cytoplasmic and nuclear eventsassociated with Fas-mediated apoptosis in vivo”, (1998) Proc. Natl.Acad. Sci. USA 95:618-23; U.S. Pat. No. 6,372,907), apoptosis (Vermes,“A novel assay for apoptosis. Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluoresceinlabeled Annexin V” (1995) J. Immunol. Methods 184:39-51) andcytotoxicity assays. Fluorometric microvolume assay technology can beused to identify the up or down regulation by a molecule that istargeted to the cell surface (Swartzman, “A homogeneous and multiplexedimmunoassay for high-throughput screening using fluorometric microvolumeassay technology”, (1999) Anal. Biochem. 271:143-51).

Labeled immunoglobulin conjugates of the invention are useful as imagingbiomarkers and probes by the various methods and techniques ofbiomedical and molecular imaging such as: (i) MRI (magnetic resonanceimaging); (ii) MicroCT (computerized tomography); (iii) SPECT (singlephoton emission computed tomography); (iv) PET (positron emissiontomography) Chen et al (2004) Bioconjugate Chem. 15:41-49; (v)bioluminescence; (vi) fluorescence; and (vii) ultrasoundImmunoscintigraphy is an imaging procedure in which antibodies labeledwith radioactive substances are administered to an animal or humanpatient and a picture is taken of sites in the body where the antibodylocalizes (U.S. Pat. No. 6,528,624). Imaging biomarkers may beobjectively measured and evaluated as an indicator of normal biologicalprocesses, pathogenic processes, or pharmacological responses to atherapeutic intervention. Biomarkers may be of several types: Type 0 arenatural history markers of a disease and correlate longitudinally withknown clinical indices, e.g. MRI assessment of synovial inflammation inrheumatoid arthritis; Type I markers capture the effect of anintervention in accordance with a mechanism-of-action, even though themechanism may not be associated with clinical outcome; Type II markersfunction as surrogate endpoints where the change in, or signal from, thebiomarker predicts a clinical benefit to “validate” the targetedresponse, such as measured bone erosion in rheumatoid arthritis by CT.Imaging biomarkers thus can provide pharmacodynamic (PD) therapeuticinformation about: (i) expression of a target protein, (ii) binding of atherapeutic to the target protein, i.e. selectivity, and (iii) clearanceand half-life pharmacokinetic data. Advantages of in vivo imagingbiomarkers relative to lab-based biomarkers include: non-invasivetreatment, quantifiable, whole body assessment, repetitive dosing andassessment, i.e. multiple time points, and potentially transferableeffects from preclinical (small animal) to clinical (human) results. Forsome applications, bioimaging supplants or minimizes the number ofanimal experiments in preclinical studies.

Radionuclide imaging labels include radionuclides such as ³H, ¹¹C, ¹⁴C,¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or ²¹³Bi. The radionuclide metal ion can becomplexed with a chelating linker such as DOTA. Linker reagents such asDOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) can be prepared by thereaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka)activated with isopropylchloroformate (Aldrich), following the procedureof Axworthy et al (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807).DOTA-maleimide reagents react with the free cysteine amino acids of themodified immunoglobulins and provide a metal complexing ligand on theantibody (Lewis et al (1998) Bioconj. Chem. 9:72-86). Chelating linkerlabelling reagents such as DOTA-NHS(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) are commercially available (Macrocyclics,Dallas, Tex.). Receptor target imaging with radionuclide labeledantibodies can provide a marker of pathway activation by detection andquantitation of progressive accumulation of antibodies in tumor tissue(Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210). Theconjugated radio-metals may remain intracellular following lysosomaldegradation.

Peptide labelling methods are well known. See Haugland, 2003, MolecularProbes Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, (1997)Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Glazer et al (1975) ChemicalModification of Proteins. Laboratory Techniques in Biochemistry andMolecular Biology (T. S. Work and E. Work, Eds.) American ElsevierPublishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984)Chemical Reagents for Protein Modification, Vols. I and II, CRC Press,New York; Pfleiderer, G. (1985) “Chemical Modification of Proteins”,Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter,Berlin and New York; and Wong (1991) Chemistry of Protein Conjugationand Cross-linking, CRC Press, Boca Raton, Fla.); De Leon-Rodriguez et al(2004) Chem. Eur. J. 10:1149-1155; Lewis et al (2001) Bioconjugate Chem.12:320-324; Li et al (2002) Bioconjugate Chem. 13:110-115; Mier et al(2005) Bioconjugate Chem. 16:240-237.

Peptides and proteins labeled with two moieties, a fluorescent reporterand quencher in sufficient proximity undergo fluorescence resonanceenergy transfer (FRET). Reporter groups are typically fluorescent dyesthat are excited by light at a certain wavelength and transfer energy toan acceptor, or quencher, group, with the appropriate Stokes shift foremission at maximal brightness. Fluorescent dyes include molecules withextended aromaticity, such as fluorescein and rhodamine, and theirderivatives. The fluorescent reporter may be partially or significantlyquenched by the quencher moiety in an intact peptide. Upon cleavage ofthe peptide by a peptidase or protease, a detectable increase influorescence may be measured (Knight, C. (1995) “Fluorimetric Assays ofProteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18-34).

The labeled antibodies of the invention may also be used as an affinitypurification agent. In this process, the labeled antibody is immobilizedon a solid phase such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody is contacted with asample containing the antigen to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the antigen to be purified, which is boundto the immobilized polypeptide variant. Finally, the support is washedwith another suitable solvent, such as glycine buffer, pH 5.0, that willrelease the antigen from the polypeptide variant.

Labelling reagents typically bear reactive functionality which may react(i) directly with a cysteine thiol of a modified immunoglobulin to formthe labeled antibody, (ii) with a linker reagent to form a linker-labelintermediate, or (iii) with a linker antibody to form the labeledantibody. Reactive functionality of labelling reagents include:maleimide, haloacetyl, iodoacetamide succinimidyl ester (e.g. NHS,N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride,2,6-dichlorotriazinyl, pentafluorophenyl ester, and phosphoramidite,although other functional groups can also be used.

An exemplary reactive functional group is N-hydroxysuccinimidyl ester(NHS) of a carboxyl group substituent of a detectable label, e.g. biotinor a fluorescent dye. The NHS ester of the label may be preformed,isolated, purified, and/or characterized, or it may be formed in situand reacted with a nucleophilic group of an antibody. Typically, thecarboxyl form of the label is activated by reacting with somecombination of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU(O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), an activator, such as 1-hydroxybenzotriazole(HOBt), and N-hydroxysuccinimide to give the NHS ester of the label. Insome cases, the label and the antibody may be coupled by in situactivation of the label and reaction with the antibody to form thelabel-antibody conjugate in one step. Other activating and couplingreagents include TBTU(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH(N,N′,N″,N′″-tetramethyluronium2-fluoro-hexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl sulfonylhalides, e.g. triisopropylbenzenesulfonyl chloride.

It is accordingly an object of the present invention to provide uses ofthe immunoglobulin conjugates as discussed in paragraph [0007] and anyand all combinations of their embodiments as a diagnostic tool.

It is accordingly an object of the present invention to provide uses ofthe immunoglobulin conjugates as discussed in paragraph [0007] and anyand all combinations of their embodiments as a standard for highmolecular weight proteins.

Immunoglobulin Polymer Conjugates

In further embodiments, the present invention also contemplatesimmunoglobulin conjugates, in which an immunoglobulin is linked with apolymer. Typically, the polymer is water soluble so that animmunoglobulin component does not precipitate in an aqueous environment,such as a physiological environment. An example of a suitable polymer isone that has been modified to have a single reactive group, such as anactive ester for acylation, or an aldehyde for alkylation. In this way,the degree of polymerization can be controlled. An example of a reactivealdehyde is polyethylene glycol propionaldehyde, or mono-(C₁-C₁₀)alkoxy, or aryloxy derivatives thereof (see, for example, Harris, etal., U.S. Pat. No. 5,252,714). The polymer may be branched orunbranched. Moreover, a mixture of polymers can be used to produceconjugates with antibody components.

Suitable water-soluble polymers include, without limitation,polyethylene glycol (PEG), monomethoxy-PEG, mono-(C₁-C₁₀)alkoxy-PEG,aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEGpropionaldehyde, bis-succinimidyl carbonate PEG, propylene glycolhomopolymers, a polypropylene oxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran,cellulose, or other carbohydrate-based polymers. Suitable PEG may have amolecular weight from about 600 to about 60,000, including, for example,5,000, 12,000, 20,000 and 25,000. A conjugate can also comprise amixture of such water-soluble polymers.

As an illustration, a polyalkyl oxide moiety can be attached to theN-terminus of an immunoglobulin component. PEG is one suitable polyalkyloxide. For example, an immunoglobulin can be modified with PEG, aprocess known as “PEGylation.” PEGylation of an immunoglobulin can becarried out by any of the PEGylation reactions known in the art (see,for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)). For example, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, immunoglobulin conjugatesare formed by condensing activated PEG, in which a terminal hydroxy oramino group of PEG has been replaced by an activated linker (see, forexample, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with an immunoglobulin. An example of an activated PEGester is PEG esterified to N-hydroxysuccinimide. As used herein, theterm “acylation” includes the following types of linkages between animmunoglobulin and a water soluble polymer: amide, carbamate, urethane,and the like. Methods for preparing PEGylated anti-B CMA-TACIimmunoglobulins by acylation will typically comprise the steps of (a)reacting an immunoglobulin with PEG (such as a reactive ester of analdehyde derivative of PEG) under conditions whereby one or more PEGgroups attach to the immunoglobulin, and (b) obtaining the reactionproduct(s). Generally, the optimal reaction conditions for acylationreactions will be determined based upon known parameters and desiredresults. For example, the larger the ratio of PEG: antibody component,the greater the percentage of polyPEGylated antibody component product.

The product of PEGylation by acylation is typically a polyPEGylatedimmunoglobulin product, wherein the lysine s-amino groups are PEGylatedvia an acyl linking group. An example of a connecting linkage is anamide. Typically, the resulting immunoglobulin component will be atleast 95% mono-, di-, or tri-pegylated, although some species withhigher degrees of PEGylation may be formed depending upon the reactionconditions. PEGylated species can be separated from unconjugatedimmunoglobulin components using standard purification methods, such asdialysis, ultrafiltration, ion exchange chromatography, affinitychromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with an immunoglobulin component in the presence of areducing agent. PEG groups can be attached to the polypeptide via a—CH₂—NH group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the s-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer antibody component conjugate molecule can comprise the stepsof: (a) reacting an antibody component with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amino group at the amino terminus of the antibodycomponent, and (b) obtaining the reaction product(s). The reducing agentused for reductive alkylation should be stable in aqueous solution andpreferably be able to reduce only the Schiff base formed in the initialprocess of reductive alkylation. Preferred reducing agents includesodium borohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer immunoglobulinconjugates, the reductive alkylation reaction conditions are those whichpermit the selective attachment of the water soluble polymer moiety tothe N-terminus of the immunoglobulin. Such reaction conditions generallyprovide for pKa differences between the lysine amino groups and theα-amino group at the N-terminus The pH also affects the ratio of polymerto protein to be used. In general, if the pH is lower, a larger excessof polymer to protein will be desired because the less reactive theN-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer: antibody component neednot be as large because more reactive groups are available. Typically,the pH will fall within the range of 3 to 9, or 3 to 6.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal Biochem. 247:434 (1997)).

Immunoglobulin Drug Conjugates

In further embodiments, the present invention includes immunoglobulinconjugates in which an immunoglobulin is conjugated to a drug orcytotoxic moiety. The drug moiety of the immunoglobulin drug conjugatesmay, for example, include any compound, moiety or group which has acytotoxic or cytostatic effect. Drug moieties include, withoutlimitation: (i) chemotherapeutic agents, which may function asmicrotubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors,or DNA intercalators; (ii) protein toxins, which may functionenzymatically; and (iii) radioisotopes.

Exemplary drug moieties include, but are not limited to, a maytansinoid,an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicinand other enediyne antibiotics, a taxane, an anthracycline, andstereoisomers, isosteres, analogs or derivatives thereof.

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PROC. NAT. ACAD. SCI. (USA)99:7968-7973), or maytansinol and maytansinol analogues preparedsynthetically according to known methods.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂ OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-I 5-hydroxy/acyloxy (U.S. Pat.No. 4,364,866) (prepared by the conversion of maytansinol byStreptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)(isolated from Trewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos.4,362,663 and 4,322,348) (prepared by the demethylation of maytansinolby Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared bythe titanium trichloride/LAH reduction of maytansinol). Many positionson maytansine compounds are known to be useful as the linkage position,depending upon the type of link. For example, for forming an esterlinkage, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with a hydroxylgroup and the C-20 position having a hydroxyl group are all suitable.

Maytansine compounds inhibit cell proliferation by inhibiting theformation of microtubules during mitosis through inhibition ofpolymerization of the microtubulin protein, tubulin (Remillard et al(1975) Science 189:1002-1005). Maytansine and maytansinoids are highlycytotoxic but their clinical use in cancer therapy has been greatlylimited by their severe systemic side-effects primarily attributed totheir poor selectivity for tumors. Clinical trials with maytansine hadbeen discontinued due to serious adverse effects on the central nervoussystem and gastrointestinal system (Issel et al (1978) Can. Treatment.Rev. 5:199-207).

Maytansinoid drug moieties are attractive drug moieties inimmunoglobulin drug conjugates because they are: (i) relativelyaccessible to prepare by fermentation or chemical modification,derivatization of fermentation products, (ii) amenable to derivatizationwith functional groups suitable for conjugation through thenon-disulfide linkers to antibodies, (iii) stable in plasma, and (iv)effective against a variety of tumor cell lines (US 2005/0169933; WO2005/037992; U.S. Pat. No. 5,208,020).

As with other drug moieties, all stereoisomers of the maytansinoid drugmoiety are contemplated for the compounds of the invention.

The drug moiety of the immunoglobulin drug conjugates also includedolastatins and their peptidic analogs and derivatives, the auristatins(U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins havebeen shown to interfere with microtubule dynamics, GTP hydrolysis, andnuclear and cellular division (Woyke et al (2001) Antimicrob. Agents andChemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.Agents Chemother. 42:2961-2965). Various forms of a dolastatin orauristatin drug moiety may be covalently attached to an antibody throughthe N (amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172; Doronina et al (2003) Nature Biotechnology21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in: WO2005/081711; Senter et al, Proceedings of the American Association forCancer Research, Volume 45, Abstract Number 623, presented Mar. 28,2004, the disclosure of each which are expressly incorporated byreference in their entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Luibke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry.

The drug moiety includes calicheamicin, and analogs and derivativesthereof. The calicheamicin family of antibiotics are capable ofproducing double-stranded DNA breaks at sub-picomolar concentrations.For the preparation of conjugates of the calicheamicin family, see U.S.Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701,5,770,710; 5,773,001; 5,877,296. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al Cancer Research53:3336-3342 (1993), Lode et al Cancer Research 58:2925-2928 (1998).

Protein toxins include, for example, diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain (Vitetta et al (1987) Science, 238:1098),abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes (WO 93/21232).

Therapeutic radioisotopes include, for example, ³²P, ³³P, ⁹⁰Y, ¹²⁵I,¹³¹I, ¹³¹In, ¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, ²¹²Pb, and radioactiveisotopes of Lu.

The radioisotope or other labels may be incorporated in the conjugate inknown ways (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80:49-57; “Monoclonal Antibodies in Immunoscintigraphy” Chatal, CRC Press1989). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of a radionuclide to the antibody (WO 94/11026).

Linkers

In certain embodiments of the present invention, the immunoglobulinconjugate includes a linker molecule having at least two reactive sites.One reactive site is bound to the substituted cysteine residue of theimmunoglobulin, and the other reactive site is bound to an atom ormolecule. A “linker” is a bifunctional or multifunctional moiety whichcan be used to link one or more drug moieties and an immunoglobulin unitto form immunoglobulin conjugates. Immunoglobulin conjugates can beconveniently prepared using a linker having reactive functionality forbinding to the drug or other molecule and to the immunoglobulin. Acysteine thiol of a modified immunoglobulin with a substitution tocysteine can form a bond with a functional group of a linker reagent, adrug moiety or drug-linker intermediate.

In one aspect, a linker has a reactive site which has an electrophilicgroup that is reactive to a nucleophilic cysteine present on anantibody. The cysteine thiol of the antibody is reactive with anelectrophilic group on a linker and forms a covalent bond to a linker.Useful electrophilic groups include, but are not limited to, maleimideand haloacetamide groups.

Modified immunoglobulins of the invention react with linker reagents ordrug-linker intermediates, with electrophilic functional groups such asmaleimide or α-halo carbonyl, according to the conjugation method atpage 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773.

The linker may comprise amino acid residues. The amino acid unit, whenpresent, links the immunoglobulin to the drug moiety of theimmunoglobulin drug conjugates of the invention.

The amino acid linker may be, for example, a dipeptide, tripeptide,tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide,nonapeptide, decapeptide, undecapeptide or dodecapeptide unit Amino acidresidues which comprise the amino acid unit include those occurringnaturally, as well as minor amino acids and non-naturally occurringamino acid analogs, such as citrulline.

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the drug moiety.

In another embodiment, the linker may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers canincrease the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the immunoglobulin drug conjugate. Thus, wherea modified immunoglobulin bears only one reactive cysteine thiol group,a multitude of drug moieties may be attached through a dendritic linker.

In another embodiment, the linker may be substituted with groups whichmodulated solubility or reactivity. For example, a charged substituentsuch as sulfonate (—SO₃ ⁻) or ammonium, may increase water solubility ofthe reagent and facilitate the coupling reaction of the linker reagentwith the immunoglobulin or the drug moiety, or facilitate the couplingreaction of the immunoglobulin-linker intermediate with the drug moiety,or the drug-linker intermediate with the immunoglobulin, depending onthe synthetic route employed to prepare the immunoglobulin conjugate.

The compounds of the invention expressly contemplate, but are notlimited to, immunoglobulin conjugates prepared with linker reagents:BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃, and BM(PEO)₄, which arecommercially available from Pierce Biotechnology, Inc., Customer ServiceDepartment, P.O. Box 117, Rockford, Ill. 61105 U.S.A, U.S.A1-800-874-3723, International +815-968-0747. See pages 467-498,2003-2004 Applications Handbook and Catalog. Bis-maleimide reagentsallow the attachment of the thiol group of a cysteine to athiol-containing drug moiety, label, or linker intermediate, in asequential or concurrent fashion. Other functional groups besidesmaleimide, which are reactive with a thiol group of a cysteine, drugmoiety, label, or linker intermediate include, without limitation,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

It is accordingly an object of the present invention to provideimmunoglobulin conjugates comprising an immunoglobulin having at leastone mutation at a residue selected from the group consisting of7(V_(H)), 20(V_(L)), 22(V_(L)), 25(V_(H)), 125(C_(H1)), 248(C_(H2)),254(C_(H2)), 286(C_(H2)), 298(C_(H2)), and 326(C_(H2)), wherein the atleast one mutation is a substitution with a cysteine residue, and anatom or molecule, wherein the atom or molecule is conjugated to thecysteine residue. In certain embodiments, the at least one mutation isat a residue selected from the group consisting of 7(V_(H)), 20(V_(L)),22(V_(L)) and 125(C_(H1)). In certain embodiments, the at least onemutation is at a residue selected from the group consisting of248(C_(H2)) and 326(CH₂). In certain embodiments, the at least onemutation is at a residue selected from the group consisting of 25(V_(H))and 286(C_(H2)). In certain embodiments, the at least one mutation is atresidue selected from the group consisting of 254(C_(H2)) and298(V_(H)). In certain embodiments that may be combined with thepreceding embodiments, the immunoglobulin is selected from the groupcomprising IgG1, IgG2, IgG3, and IgG4. In certain embodiments that maybe combined with the preceding embodiments, the immunoglobulin comprisesan IgG1. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(H1)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(H2)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(H3)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human C_(L)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human V_(H)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate comprises a human V_(L)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin conjugate further comprises a linkermolecule having at least two reactive sites, wherein a first reactivesite is bound to the cysteine residue of the immunoglobulin and a secondreactive site is bound to the atom or molecule. In certain embodimentsthat may be combined with the preceding embodiments having a linkermolecule, the linker molecule is selected from the group consisting of ahydrazone, a disulfide, a peptide, a chelating agent, and a maleimide.In certain embodiments that may be combined with the precedingembodiments, the atom or molecule is selected from the group consistingof a radionuclide, a chemotherapeutic agent, a microbial toxin, a planttoxin, a polymer, a carbohydrate, a cytokine, a fluorescent label, aluminescent label, an enzyme-substrate label, an enzyme, a peptide, apeptidomimetic, a nucleotide, an siRNA, a microRNA, an RNA mimetic, andan aptamer. In certain embodiments that may be combined with thepreceding embodiments, the atom or molecule is selected from the groupconsisting of ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹⁷⁷Lu, ²¹³Bi, ²¹¹At, a calicheamicin, aduocarmycin, a maytanisoid, an auristatin, an anthracyclin, Pseudomonasexotoxin A, Diptheria toxin, ricin, polyethylene glycol, hydroxyethylstarch, and a mannosyl residue. In certain embodiments that may becombined with the preceding embodiments, the atom or molecule reducesthe immunogenicity of the unmutated immunoglobulin. In certainembodiments that may be combined with the preceding embodiments, theatom or molecule increases the immunogenicity of the unmutatedimmunoglobulin. In certain embodiments that may be combined with thepreceding embodiments, the immunoglobulin conjugate further comprises anantigen binding activity and the activity is at least eighty percent, atleast ninety percent, at least one hundred percent, at least one hundredten percent, at least one hundred twenty percent, or at least onehundred thirty percent of the antigen binding activity of the unmutatedimmunoglobulin.

It is accordingly an object of the present invention to provide modifiedor isolated immunoglobulins comprising at least one mutation at aresidue selected from the group consisting of 7(V_(H)), 20(V_(L)),22(V_(L)), 25(V_(H)), 125(C_(H1)), 248(C_(H2)), 254(C_(H2)),286(C_(H2)), and 326(C_(H2)), wherein the at least one mutation is asubstitution with a cysteine residue. In certain embodiments the atleast one mutation is at a residue selected from the group consisting of7(V_(H)), 20(V_(L)), 22(V_(L)) and 125(C_(H1)). In certain embodimentsthe at least one mutation is at a residue selected from the groupconsisting of 248(C_(H2)) and 326(C_(H2)). In certain embodiments the atleast one mutation is at a residue selected from the group consisting of25(V_(H)) and 286(C_(H2)). In certain embodiments the at least onemutation is at residue 254(C_(H2)). In certain embodiments that may becombined with the preceding embodiments, the immunoglobulin is selectedfrom the group comprising IgG1, IgG2, IgG3, and IgG4. In certainembodiments that may be combined with the preceding embodiments, theimmunoglobulin comprises an IgG1. In certain embodiments that may becombined with the preceding embodiments, the modified or isolatedimmunoglobulin comprises a human C_(H1) domain. In certain embodimentsthat may be combined with the preceding embodiments, the modified orisolated immunoglobulin comprises a human C_(H2) domain. In certainembodiments that may be combined with the preceding embodiments, themodified or isolated immunoglobulin comprises a human C_(H3) domain. Incertain embodiments that may be combined with the preceding embodiments,the modified or isolated immunoglobulin comprises a human C_(L) domain.In certain embodiments that may be combined with the precedingembodiments, the modified or isolated immunoglobulin comprises a humanV_(H) domain. In certain embodiments that may be combined with thepreceding embodiments, the modified or isolated immunoglobulin comprisesa human V_(L) domain. In certain embodiments that may be combined withthe preceding embodiments, the immunoglobulin further comprises anantigen binding activity and the activity is at least eighty percent, atleast ninety percent, at least one hundred percent, at least one hundredten percent, at least one hundred twenty percent, or at least onehundred thirty percent of the antigen binding activity of the unmutatedimmunoglobulin.

Preparation of Immunoglobulin Drug Conjugates

In one aspect, the present invention includes methods of producingimmunoglobulin conjugates. The immunoglobulin drug conjugate may beprepared by several routes, employing organic chemistry reactions,conditions, and reagents known to those skilled in the art, including:(1) reaction of a cysteine group of a modified immunoglobulin with alinker reagent, to form an immunoglobulin-linker intermediate, via acovalent bond, followed by reaction with an activated drug moiety; and(2) reaction of a nucleophilic group of a drug moiety with a linkerreagent, to form a drug-linker intermediate, via a covalent bond,followed by reaction with a cysteine group of a modified immunoglobulin.Conjugation methods (1) and (2) may be employed with a variety ofmodified immunoglobulins, drug moieties, and linkers to prepare theimmunoglobulin drug conjugates of the invention.

Antibody cysteine thiol groups are nucleophilic and capable of reactingto form covalent bonds with electrophilic groups on linker reagents anddrug-linker intermediates including: (i) active esters such as NHSesters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides, such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups; and (iv) disulfides, including pyridyldisulfides, via sulfide exchange. Nucleophilic groups on a drug moietyinclude, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents.

Maytansine may, for example, be converted to May-SSCH₃, which can bereduced to the free thiol, May-SH, and reacted with a modified antibody(Chari et al (1992) Cancer Research 52:127-131) to generate amaytansinoid-antibody immunoconjugate with a disulfide linker.Antibody-maytansinoid conjugates with disulfide linkers have beenreported (WO 04/016801; U.S. Pat. No. 6,884,874; US 2004/039176 A1; WO03/068144; US 2004/001838 A1; U.S. Pat. Nos. 6,441,163, 5,208,020,5,416,064; WO 01/024763). The disulfide linker SPP is constructed withlinker reagent N-succinimidyl 4-(2-pyridylthio)pentanoate.

Under certain conditions, the modified immunoglobulins may be madereactive for conjugation with linker reagents by treatment with areducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.) or otherreducing agents known to one of skill in the art.

It is accordingly an object of the present invention to provide methodsof producing immunoglobulin conjugates by providing modified or isolatedimmunoglobulins as discussed in paragraphs [0008] or [0019] and any andall combinations of their embodiments, reducing the one or moresubstituted cysteine residues with a reducing agent to form reducedcysteine residues, and incubating the immunoglobulin with an atom ormolecule, wherein the atom or molecule is reactive with the reducedcysteine residues, to form an immunoglobulin conjugate.

Spatial-Aggregation-Propensity

In one aspect, the invention herein relates to methods for selectingresidues on a protein surface to mutate to cysteine and for reducingcross-linking of a modified immunoglobulin or immunoglobulin conjugate.The invention may be applied to generate immunoglobulins andimmunoglobulin conjugates with reduced propensity for cross-linking,i.e., the immunoglobulin or immunoglobulin conjugate in concentratedsolution remains primarily in monomeric form rather than higher orderaggregated multimers. The methods herein represent an advancement in theability of computational methods to evaluate the propensity of a proteinto cross-link. In particular, the methods are based, at least in part,on the calculation of the SAA (Solvent Accessible Area), which is knownin the art for characterizing the surface of a protein. SAA gives thesurface area of each amino acid or protein structure that is in contactwith the solvent. SAA may be typically calculated by computing the locusof the center of a probe sphere as it rolls over the protein surface,i.e., the surface of a protein structural model. The probe sphere hasthe same radius as that of a water molecule, R=1.4 Å. Alternativemethods of calculating SAA, described below, are known in the art andare compatible with the methods described herein. Although SAA is quiteuseful to characterize the protein surface, it was not found to beadequate to characterize the hydrophobic patches on the protein surfacethat are potentially aggregation prone because of the followingshortcomings,

-   1. SAA doesn't distinguish between hydrophobic and hydrophilic    regions-   2. SAA is not directly proportional to a residue's hydrophobicity    (for example, MET has more surface area than LEU but is less    hydrophobic)-   3. SAA doesn't indicate whether several hydrophobic residues are    close-by and thus could enhance the hydrophobicity of a certain    region. These residues could be close-by either in primary sequence    or in the tertiary structure even though they are far in primary    sequence. Either way, they could enhance the hydrophobicity of a    certain patch on the antibody surface.

One measure which is described herein, the Effective-SAA, is generatedby calculating the hydrophobicity of the fraction of the amino acidwhich is exposed according to the formula below:

${{Effective} - {SAA}} = {\frac{SAA}{{SAA}_{{fully}\text{-}{exposed}}} \times {Residue}\mspace{14mu} {hydrophobicity}}$

A further embodiment of the Effective-SAA further comprises summing theEffective-SAA over at least two, at least three, at least four, at leastfive or at least six, (e.g., two, three, four, five, six, etc.) aminoacid residues which are adjacent in the primary protein sequence.Although the Effective-SAA represents an improvement over the basic SAA,it nevertheless lacks the ability to fully account for the structure ofthe folded protein and for the fact that amino acids which are notadjacent in the protein sequence may be in proximity to one another inthe folded secondary, tertiary, or quaternary structure of a protein.Such protein folds may form aggregation prone regions which do notappear in the primary structure alone, or which may only be detected bymore robustly analyzing the folded protein structure.

The present invention provides a new, more advanced measure, called theSpatial-Aggregation-Propensity, which will highlight the effectivehydrophobicity of a certain patch or region on the protein surface. TheSpatial-Aggregation-Propensity is calculated for defined spatial regionson or near the atoms of a protein structural model.

In this context, a “defined spatial region” is a three-dimensional spaceor volume chosen to capture a local physical structure and/or chemicalenvironment on or near the protein structure. In a particularlypreferred embodiment the Spatial-Aggregation-Propensity is calculatedfor spherical regions with radius R centered on atoms in a protein(e.g., atoms in a protein structural model). TheSpatial-Aggregation-Propensity may also be calculated for sphericalregions with radius R centered on chemical bonds, or positioned in spacenear the structural model. Accordingly, in another embodiment the SAPmay be calculated for a defined spatial region centered near an atom,e.g., centered on a point in space which is between 1-10 Å, 1-5 Å, or1-2 Å from the center of a particular atom or chemical bond.

In certain embodiments, the chosen radius R is between 1 Å and 50 Å. Inparticular embodiments the chosen radius is at least 1 Å, at least 3 Å,at least 4 Å, at least 5 Å, at least 6 Å, at least 7 Å, at least 8 Å, atleast 9 Å, at least 10 Å, at least 11 Å, at least 12 Å, at least 15 Å,at least 20 Å, at least 25 Å, or at least 30 Å. In certain embodiments,the chosen radius is between 5 Å and 15 Å, between 5 Å and 12 Å, orbetween 5 Å and 10 Å. In specific embodiments the chosen radius is 5 Åor 10 Å.

In other embodiments, the region for which theSpatial-Aggregation-Propensity is calculated is not spherical. Thepossible shape of the region may further comprise a cube, a cylinder, acone, an elliptical spheroid, a pyramid, a hemisphere, or any othershape which may be used to enclose a portion of space. In suchembodiments, the size of the region may be chosen using measures otherthan radius, e.g., the distance from the center of the shape to a faceor vertex.

In a certain embodiment, the SAP may be used to select residues in aprotein, particularly an antibody or immunoglobulin, which may besubstituted with cysteine without increasing the protein's propensity tocross-link. The present invention is expected to streamline the processof identifying residues that can be substituted with cysteine withoutincreasing the propensity for cross-linking.

Thus, in general terms, a method for calculating theSpatial-Aggregation-Propensity for a particular atom in a proteincomprises (a) identifying one or more atoms in a structural modelrepresenting the protein, wherein the one or more atoms are within adefined spatial region centered on or near the particular atom; (b)calculating, for each of the one or more atoms in the defined spatialregion, a ratio of the solvent accessible area (SAA) of the atoms to theSAA of atoms in an identical residue which is fully exposed; (c)multiplying each ratio by the atom hydrophobicity of the one or moreatoms; and (d) summing the products of step (c); whereby the sum is theSAP for the particular atom.

In a related embodiment, the SAP may be calculated according to adifferent method comprising (a) identifying one or more amino acidresidues in a structural model representing the protein, wherein the oneor more amino acid residues have at least one atom within a definedspatial region centered on or near the particular atom; (b) calculating,for each of the identified one or more amino acid residues, a ratio ofthe solvent accessible area (SAA) of atoms in the amino acid to the SAAof atoms in an identical residue which is fully exposed; (c) multiplyingeach ratio by the hydrophobicity of the one or more amino acid residuesas determined by an amino acid hydrophobicity scale; and (d) summing theproducts of step (c); whereby the sum is the SAP for the particularatom. In preferred embodiments, the structural model is processed priorto step (a) by allowing the structural model to interact with solvent ina molecular dynamics simulation. When an amino acid is identified ashaving at least one atom within the defined spatial region, the at leastone atom may be required to be exclusively an atom in an amino acid sidechain. Alternatively it may be an atom required to be a main chain atom.

In other embodiments, this method may further comprise optionallyconducting a molecular dynamics simulation prior to step (a) andrepeating steps (a)-(d), each time conducting a further moleculardynamics simulation at a plurality of time steps, thereby producingmultiple sums as in step (d), and calculating the average of the sums;whereby the calculated average is the SAP for the particular atom.

One of skill in the art will appreciate that an embodiment of thepresent invention which employs the average of values calculated over amolecular dynamics simulation will be more computationally intensive.Such an embodiment will also, in some cases, provide a more precise orhighly resolved map of the Spatial-Aggregation-Propensity. However,experiments discussed herein have shown that the method is still highlyaccurate when the molecular dynamics averaging is not employed. In onepreferred embodiment, Spatial-Aggregation-Propensity values may becalculated for all protein structures in a database, e.g., the ProteinData Bank (PDB), thereby swiftly identifying hydrophobic residues andpatches on all known protein structures. This method allows rapidscreening of large sets of proteins to identify potential aggregationprone regions and/or protein interaction sites.

In a preferred application, the Spatial-Aggregation-Propensity isdescribed

by the following formula:

SAP_(atom)=Σ_(Simulation Average)(Σ_(atoms within R of atom)((SAA-R/SAA-fe)*atom-hb)

wherein:

-   -   1) SAA-R is SAA of side chain atoms within radius R which is        computed at each simulation snapshot. SAA is preferably        calculated in the simulation model by computing the locus of the        center of a probe sphere as it rolls over the protein surface.        The probe sphere has the same radius as that of a water        molecule, R=1.4 A. One of skill in the art will appreciate that        other methods of computing the SAA would be compatible with the        methods described here to calculate SAP. For example, the SAA        may be calculated on only amino acid side chain atoms. The SAA        may also be calculated on only amino acid main chain atoms        (i.e., those atoms of the peptide backbone and associated        hydrogens). Alternatively, the SAA may be calculated on only        amino acid main chain atoms with the exclusion of associated        hydrogens;    -   2) SAA-fe is SAA of side chain of fully exposed residue (say for        amino acid ‘X’) which is obtained, in a preferred embodiment, by        calculating the SAA of side chains of the middle residue in the        fully extended conformation of tripeptide ‘Ala-X-Ala’; and    -   3) atom-hb is Atom Hydrophobicity which is obtained as described        above using the hydrophobicity scale of Black and Mould (Black        and Mould, Anal. Biochem. 1991, 193, 72-82). The scale is        normalized such that Glycine has a hydrophobicity of zero.        Therefore, amino acids that are more hydrophobic than Glycine        are positive and less hydrophobic than Glycine are negative on        the hydrophobic scale.

A residue which is “fully exposed” is a residue, X, in the fullyextended conformation of the tripeptide Ala-X-Ala. One of skill in theart will appreciate that this arrangement is designed such that acalculation of SAA on such a residue, X, will yield the maximum solventaccessible area available. Accordingly, it is contemplated that otherresidues besides alanine may be used in the calculation without whollydisrupting or altering the results.

As described above, the methods of the present invention may be appliedto any protein structural model including an X-ray structure using thesame formula as above.

Similarly, if the X-ray structure is not available, the sameSpatial-Aggregation-Propensity parameter can be applied to the structuregenerated through homology modeling, and the SAP parameter may becalculated using the same formula as above.

In certain embodiments the Spatial-Aggregation-Propensity is calculatedfor all atoms in a protein structural model. In some embodiments, theatomistic Spatial-Aggregation-Propensity values may be averaged overeach individual protein residue, or over small groups of residues.

Uses of the SAP Methodology

In one aspect, the present invention may be used as described above toidentify hydrophobic amino acid residues, regions or patches in aprotein. Without wanting to be held to specific threshold values, atomsor amino acid residues having a Spatial-Aggregation-Propensity >0 areconsidered to be hydrophobic, or to be in an aggregation prone region.Depending on the type of protein, the particular structure, and thesolvent in which it exists, it may be desirable to identify atoms orresidues using a cutoff which is slightly below zero, e.g., by choosingatoms or residues which have a Spatial-Aggregation-Propensity of greaterthan −0.1, −0.15, −0.2, etc. Alternatively, it may be desirable toemploy a more stringent cutoff, e.g., 0, 0.05, 0.1, 0.15, 0.2, etc., inorder to choose the strongest hydrophobic atoms, residues, or patches.In addition, as the algorithm gives higher numbers to residues at thecenter of a patch, residues within 3 A, 4 A, 5 A, 7.5 A, or 10 A of theresidue meeting the cutoff can also be selected for mutation to lesshydrophobic residues to reduce aggregation. In another embodiment, itmay be advantageous simply to select atoms or residues havingSpatial-Aggregation-Propensity which is larger than atoms or residueswhich are nearby either sequentially (i.e., along the protein sequence)or, in a preferred embodiment, spatially (i.e., in the three-dimensionalstructure). One preferred method for selecting atoms or residues in ahydrophobic patch is to map the calculatedSpatial-Aggregation-Propensity values, e.g., using a color coding ornumerical coding, onto the protein structural model from which they werederived, thus visualizing differences in theSpatial-Aggregation-Propensity across the protein surface and henceallowing easy selection of hydrophobic patches or residues. In aparticularly preferred embodiment, the calculations forSpatial-Aggregation-Propensity are carried out separately using twovalues chosen for the radius, one of higher resolution, e.g., 5 A, andone of lower resolution, e.g., 10 A. In such an embodiment larger orbroader hydrophobic patches may be seen on the protein structure withthe lower resolution map. Once hydrophobic patches of interest areselected on the low resolution map, those patches may be viewed ingreater detail in the higher resolution map which may, in someembodiments, allow one of skill in the art to more easily or moreaccurately choose residues to mutate or modify. For example, whenviewing a hydrophobic patch in the higher resolution map, it may bedesirable to select for mutation the residue which has the highest SAPscore or is the most hydrophobic (e.g., the most hydrophobic residue inthe patch according to the scale of Black and Mould, Anal. Biochem.1991, 193, 72-82).

In a specific embodiment a method to identify an aggregation proneregion on a protein comprises (a) mapping onto the structural model theSAP as calculated according to any of the methods described herein foratoms in the protein; and (b) identifying a region within in the proteinhaving a plurality of atoms having a SAP >0; wherein the aggregationprone region comprises the amino acids comprising said plurality ofatoms. In such an embodiment the SAP may be calculated for all the atomsin a protein or a portion of the atoms. It is contemplated that one mayonly calculate the SAP for particular residues or groups of residueswhich are of interest.

In a similar embodiment, it may be informative to plot the SAP scores ofthe atoms (or the SAP score as averaged over amino acid residues). Sucha plot showing the SAP score along the atoms or residues of a proteinallows the easy identification of peaks, which may indicate candidatesfor replacement. In a particularly preferred embodiment the SAP scoresalong the atoms or residues in the protein are plotted in a graph andthe Area Under the Curve (AUC) is calculated for peaks in the graph. Insuch an embodiment, peaks with a larger AUC represent larger or morehydrophobic aggregation prone regions. In particular embodiments it willbe desirable to select for replacement one or more residues which areidentified as existing in a peak, or, more preferably, in a peak with alarge AUC.

In particular embodiments the present invention may be used to select aresidue of an immunoglobulin for mutation to cysteine. As used herein,the SAP value of a first amino acid residue on the surface of animmunoglobulin is calculated. If the SAP value is equal to or in betweenthe values of 0 and −0.11, the first residue is selected for mutation tocysteine. In a further embodiment, the SAP values of a plurality ofresidues of the immunoglobulin within immediate proximity of the firstresidue are calculated. If the plurality of residues has SAP values ofless than 0, the first residue is selected for mutation to cysteine.

Immunoglobulin variants may be made by any method known in the artincluding site directed mutagenesis and other recombinant DNAtechnology, e.g., see U.S. Pat. Nos. 5,284,760; 5,556,747; 5,789,166;6,878,531, 5,932,419; and, 6,391,548.

In particular embodiments the present invention may be used to make animmunoglobulin variant which can be conjugated to an atom or molecule byreplacing at least one amino acid residue exposed on the surface of theimmunoglobulin identified by any of the methods described herein with anatural amino acid residue, a modified amino acid residue, an unusualamino acid residue, an unnatural amino acid residue, or an amino acidanalog or derivative which can be used for conjugating theimmunoglobulin to an atom or molecule. In preferred embodiments, theamino acid residue exposed on the surface of the immunoglobulin isreplaced with cysteine. In other embodiments, the amino acid residue isreplaced with lysine, aspartate, or pyrorlysine.

The synthesis of unnatural amino acids is known to those of skill in theart, and is further described, e.g., in U.S. Patent Publication No.2003-0082575. In general, any method known in the art to synthesize orincorporate unnatural, modified, or unusual amino acids into proteinsmay be employed including, but not limited to those methods described orreferenced in the publications Liao J. Biotechnol Prog. 2007January-February; 23(1):28-31; Rajesh, and Iqbal. Curr Pharm Biotechnol.2006 August; 7(4):247-59; Cardillo et al. Mini Rev Med Chem. 2006 March;6(3):293-304; Wang et al. Annu Rev Biophys Biomol Struct. 2006;35:225-49; Chakraborty et al., Glycoconj J. 2005 March; 22(3):83-93. Asa further example, the Ambrx ReCODE™ technology may be employed todevelop and incorporate unnatural amino acids, or unusual amino acidsinto proteins as indicated by the methods described herein.

Immunoglobulin variants and immunoglobulin conjugates according to theinvention can exhibit enhanced or improved stability as determined, forexample, by non-reducing SDS-PAGE.

It is accordingly an object of the present invention to provide isolatedor recombinant polynucleotides that encode modified immunoglobulins asdiscussed in paragraphs [0008] and [0019] and any and all combinationsof their embodiments. In certain embodiments, the polynucleotide is in avector. In certain embodiments, the vector is an expression vector. Incertain embodiments that may be combined with the preceding embodiments,an inducible promoter is operably linked to the polynucleotide. Anotheraspect includes host cells with the vector of either of the precedingembodiments. In certain embodiments, the host cells are capable ofexpressing the immunoglobulin encoded by the polynucleotide.

It is accordingly an object of the present invention to provide methodsof producing an immunoglobulin with a reduced propensity forcross-linking comprising providing a culture medium comprising the hostcell of the preceding paragraph and placing the culture medium inconditions under which the immunoglobulin is expressed. In certainembodiments, the methods include an additional step of isolating theimmunoglobulin expressed.

It is accordingly an object of the present invention to provide methodsfor selecting a residue of an immunoglobulin for mutation to cysteinecomprising calculating the Spatial-Aggregation-Propensity of a firstamino acid residue on the surface of the immunoglobulin, calculating theSpatial-Aggregation-Propensities of a plurality of residues of theimmunoglobulin within immediate proximity of the first residue, andselecting the first amino acid residue for mutation to cysteine if theSpatial-Aggregation-Propensity of the first amino acid residue is equalto or in between the values of 0 and −0.11 and if the plurality ofresidues have Spatial-Aggregation-Propensities of less than 0. Incertain embodiments, the plurality of residues is within 15 Å of thefirst residue. In certain embodiments, the plurality of residues iswithin 10 Å of the first residue. In certain embodiments, the pluralityof residues is within 7.5 Å of the first residue. In certainembodiments, the plurality of residues is within 5 Å of the firstresidue. In certain embodiments that may be combined with the precedingembodiments, calculating the Spatial-Aggregation-Propensity of a residuecomprises calculating the Spatial-Aggregation-Propensity for a sphericalregion with a radius centered on an atom in the residue. In certainembodiments, the radius of the spherical region is at least 5 Å.

In some embodiments, the invention further relates to computer code fordetermining SAP according to the methods of the invention. In otherembodiments, the invention relates to a computer, a supercomputer, orcluster of computers dedicated to performing the methods of theinvention. In yet another aspect, the invention provides a web-based,server based, or internet based service for selecting residues of aprotein to mutate to cysteine, the service comprising accepting dataabout a protein (e.g., a protein structural model) from a user (e.g.,over the internet) or retrieving such data from a database such that theservice provider can generate, retrieve, or access a static structure ofthe protein, optionally including molecular dynamics modeling of theprotein to provide a dynamic structure of the protein, determining SAPfor atoms or residues of the protein based on the static or dynamicstructure so generated, and returning the SAP data, for example, as astructural model mapped with said SAP data by the service provider, to auser. In some embodiments, the user is a person. In other embodimentsthe user is a computer system or automated computer algorithm.

In some embodiments the present invention proves an SAP calculationsystem comprising: a web server for providing a web service forcalculating SAP to a user terminal through the Internet; a database forstoring general information on the calculation method, amino acidhydrophobicity, etc., and a calculation server for performing the SAPcalculation based on information in the database and informationprovided or transmitted through the internet by the user.

In some embodiments, the web server and the calculation server are thesame computer system. In some embodiments the computer system is asupercomputer, a cluster computer, or a single workstation or server. Ina related embodiment the web server of the SAP calculation systemfurther comprises a controller for controlling the entire operation, anetwork connection unit for connection to the Internet, and a webservice unit for providing a web service for calculating SAP to the userterminal connected through the Internet.

In addition, embodiments of the present invention further relate tocomputer storage products with a computer readable medium that containprogram code for performing various computer-implemented operations,e.g., calculating the SAP for a structural model, calculating SAA,calculating effective-SAA, manipulating structural models, implementingmolecular dynamics simulations, organizing and storing relevant data, orperforming other operations described herein. The computer-readablemedium is any data storage device that can store data which canthereafter be read by a computer system. Examples of computer-readablemedia include, but are not limited to hard disks, floppy disks, flashdrives, optical discs (e.g., CDs, DVDs, HD-DVDs, Blu-Ray discs, etc.)and specially configured hardware devices such as application specificintegrated circuits (ASICs) or programmable logic devices (PLDs). Thecomputer-readable medium can also be distributed as a data signalembodied in a carrier wave over a network of coupled computer systems sothat the computer readable code is stored and executed in a distributedfashion. It will be appreciated by those skilled in the art that theabove described hardware and software elements are of standard designand construction. The computer, internet, server, and service relatedembodiments described above may further apply to the SAA and theeffective-SAA as well as SAP.

Pharmaceutical Compositions Containing Immunoglobulins andImmunoglobulin Conjugates

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or more immunoglobulinconjugates produced by the methods of the invention, formulated togetherwith a pharmaceutically acceptable carrier. Pharmaceutical compositionsof the invention also can be administered in combination therapy, i.e.,combined with other agents. For example, the combination therapy caninclude an immunoglobulin conjugate of the present invention combinedwith at least one other anti-cancer agent.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., theimmunoglobulin or variant thereof of the invention, may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The pharmaceutical compositions of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Exemplary formulations comprise at least one immunoglobulin conjugate ofthe invention and can comprise lower concentrations of stabilizingagents which can, in addition to the methods disclosed herein, be usedto prevent or diminish cross-linking of an immunoglobulin. Accordingly,conventional methods used to prevent cross-linking may be employed inthe development of pharmaceutical compositions containing immunoglobulinconjugates produced by the methods of the present invention. Forexample, a variety of stabilizing or disaggregating compounds may beincluded in pharmaceutical compositions of the invention depending ontheir intended use and their biological toxicity. Such stabilizingcompounds may include, for example, cyclodextrin and its derivatives(U.S. Pat. No. 5,730,969), alkylglycoside compositions (U.S. patentapplication Ser. No. 11/474,049), the use of chaperone molecules (e.g.,LEA (Goyal et al., Biochem J. 2005, 388(Pt 1):151-7; the methods of U.S.Pat. No. 5,688,651), betaine compounds (Xiao, Burn, Tolbert, BioconjugChem. 2008 May 23), surfactants (e.g., Pluronic F127, Pluronic F68,Tween 20 (Wei et al. International Journal of Pharmaceutics. 2007,338(1-2):125-132)), and the methods described in U.S. Pat. Nos.5,696,090, 5,688,651, and 6,420,122.

In addition, proteins, and in particular antibodies, are stabilized informulations using combinations of different classes of excipients,e.g., (1) disaccarides (e.g. Saccharose, Trehalose) or polyols (e.g.Sorbitol, Mannitol) act as stabilizers by preferential exclusion and arealso able to act as cryoprotectants during lyophilization, (2)surfactants (e.g. Polysorbat 80, Polysorbat 20) act by minimizinginteractions of proteins on interfaces like liquid/ice,liquid/material-surface and/or liquid/air interfaces and (3) buffers(e.g. phosphate-, citrate-, histidine) help to control and maintainformulation pH. Accordingly, such disaccharides polyols, surfactants andbuffers may be used in addition to the methods of the present inventionto further stabilize immunoglobulins and prevent their aggregation.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred per cent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

It is accordingly an object of the present invention to provide methodsfor reducing the cross-linking between surface-exposed cysteines of animmunoglobulin in a highly concentrated pharmaceutical formulation ofimmunoglobulin conjugates comprising providing an immunoglobulin,substituting a residue selected from the group consisting of 7(V_(H)),20(V_(L)), 22(V_(L)), and 125(C_(H1)) with a cysteine residue, reducingthe one or more substituted cysteine residues with a reducing agent toform reduced cysteine residues, incubating the immunoglobulin with anatom or molecule, wherein the molecule is reactive with the reducedcysteine residues, to form an immunoglobulin conjugate, and generating ahighly concentrated, liquid formulation of the immunoglobulin conjugatewherein the immunoglobulin conjugate concentration is at least 20 mg/ml,at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml. Incertain embodiments, the immunoglobulin is selected from the groupcomprising IgG1, IgG2, IgG3, and IgG4. In certain embodiments, theimmunoglobulin comprises an IgG1. In certain embodiments that may becombined with the preceding embodiments, the immunoglobulin comprises ahuman C_(H1) domain. In certain embodiments that may be combined withthe preceding embodiments, the immunoglobulin comprises a human C_(H2)domain. In certain embodiments that may be combined with the precedingembodiments, the immunoglobulin comprises a human C_(H3) domain. Incertain embodiments that may be combined with the preceding embodiments,the immunoglobulin comprises a human C_(L) domain. In certainembodiments that may be combined with the preceding embodiments, theimmunoglobulin comprises a human V_(H) domain. In certain embodimentsthat may be combined with the preceding embodiments, the immunoglobulincomprises a human V_(L) domain. In certain embodiments that may becombined with the preceding embodiments, the immunoglobulin conjugatecomprises an antigen binding activity and the activity is at leasteighty percent, at least ninety percent, at least one hundred percent,at least one hundred ten percent, at least one hundred twenty percent,or at least one hundred thirty percent of the antigen binding activityof the unmutated immunoglobulin.

It is accordingly an object of the present invention to provide modifiedimmunoglobulin formulations that can be made up of immunoglobulinconjugates as discussed in paragraph [0007] and any and all combinationsof their embodiments at a concentration of at least 10 mg/ml, at least20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, atleast 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150mg/ml. In certain embodiments, the immunoglobulin conjugate is at aconcentration of greater than the concentration at which animmunoglobulin conjugate known to have a high propensity foroligomerization forms oligomers in a concentrated, liquid solution underthe same conditions. In certain embodiments that may be combined withthe preceding embodiments, at least eighty percent, at least eighty-fivepercent, at least ninety percent, at least ninety-five percent, at leastninety-six percent, at least ninety-seven percent, at least ninety-eightpercent, or at least ninety-nine percent of the immunoglobulin conjugateis non-oligomerized monomer. In certain embodiments that may be combinedwith any of the preceding embodiments, the formulation includes apharmaceutically acceptable excipient. In certain embodiments that maybe combined with any of the preceding embodiments, the immunoglobulinformulation comprises at least eighty percent, at least eighty-fivepercent, at least ninety percent, at least ninety-five percent, at leastninety-six percent, at least ninety-seven percent, at least ninety-eightpercent, or at least ninety-nine percent of immunoglobulin conjugatethat is non-oligomerized monomer.

It is accordingly an object of the present invention to provide uses ofthe immunoglobulin conjugates as discussed in paragraph [0007] and anyand all combinations of their embodiments as a non-oligomerizingpharmaceutical active ingredient.

It is accordingly an object of the present invention to providepharmaceutical compositions that include an immunoglobulin conjugate asdiscussed in paragraph [0007] and any and all combinations of theirembodiments and a pharmaceutically acceptable excipient. In certainembodiments, the immunoglobulin is at a concentration of at least 10mg/ml, at least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least50 mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, orat least 150 mg/ml. In certain embodiments, the immunoglobulin conjugateis at a concentration of greater than the concentration at which animmunoglobulin conjugate known to have a high propensity foroligomerization forms oligomers in a concentrated, liquid solution underthe same conditions. In certain embodiments that may be combined withthe preceding embodiments, at least eighty percent, at least eighty-fivepercent, at least ninety percent, at least ninety-five percent, at leastninety-six percent, at least ninety-seven percent, at least ninety-eightpercent, or at least ninety-nine percent of the immunoglobulin conjugateis non-oligomerizing monomer. In certain embodiments that may becombined with any of the preceding embodiments, the immunoglobulinformulation comprises at least eighty percent, at least eighty-fivepercent, at least ninety percent, at least ninety-five percent, at leastninety-six percent, at least ninety-seven percent, at least ninety-eightpercent, or at least ninety-nine percent of immunoglobulin conjugatethat is non-oligomerized monomer. In certain embodiments that may becombined with preceding embodiments, the oligomerization is measured bynon-reducing SDS-PAGE.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the immunoglobulin conjugate, the dosage rangesfrom about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of thehost body weight. For example dosages can be 0.3 mg/kg body weight, 1mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once per week, once every two weeks, onceevery three weeks, once every four weeks, once a month, once every 3months or once every three to 6 months. Preferred dosage regimens for animmunoglobulin conjugate of the invention include 1 mg/kg body weight or3 mg/kg body weight via intravenous administration, with the antibodybeing given using one of the following dosing schedules: (i) every fourweeks for six dosages, then every three months; (ii) every three weeks;(iii) 3 mg/kg body weight once followed by 1 mg/kg body weight everythree weeks.

Alternatively an immunoglobulin conjugate of the invention can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the administered substance in the patient. Ingeneral, human antibodies show the longest half life, followed byhumanized antibodies, chimeric antibodies, and nonhuman antibodies. Thedosage and frequency of administration can vary depending on whether thetreatment is prophylactic or therapeutic. In prophylactic applications,a relatively low dosage is administered at relatively infrequentintervals over a long period of time. Some patients continue to receivetreatment for the rest of their lives. In therapeutic applications, arelatively high dosage at relatively short intervals is sometimesrequired until progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patient can be administered aprophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of immunoglobulin conjugate of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for binding moieties of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an immunoglobulin conjugate of the invention can beadministered via a nonparenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system.

It is accordingly an object of the present invention to provide uses ofthe immunoglobulin conjugates as discussed in paragraph [0007] and anyand all combinations of their embodiments in the preparation of amedicament comprising a highly concentrated liquid formulation whereinthe immunoglobulin conjugate concentration is at least 20 mg/ml, atleast 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml,at least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml. Incertain embodiments, the use of the medicament is for the treatment ofautoimmune diseases, immunological diseases, infectious diseases,inflammatory diseases, neurological diseases, and oncological andneoplastic diseases including cancer. In certain embodiments, the use ofthe medicament is for the treatment of congestive heart failure (CHF),vasculitis, rosacea, acne, eczema, myocarditis and other conditions ofthe myocardium, systemic lupus erythematosus, diabetes, spondylopathies,synovial fibroblasts, and bone marrow stroma; bone loss; Paget'sdisease, osteoclastoma; breast cancer; disuse osteopenia; malnutrition,periodontal disease, Gaucher's disease, Langerhans' cell histiocytosis,spinal cord injury, acute septic arthritis, osteomalacia, Cushing'ssyndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia,periodontal reconstruction, and bone fractures; sarcoidosis; osteolyticbone cancers, breast cancer, lung cancer, kidney cancer and rectalcancer; bone metastasis, bone pain management, and humoral malignanthypercalcemia, ankylosing spondylitisa and other spondyloarthropathies;transplantation rejection, viral infections, hematologic neoplasias andneoplastic-like conditions for example, Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticlymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplamacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, Langerhans cellhistocytosis, myeloid neoplasias such as acute myelogenous leukemias,including AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia,tumors of the central nervous system, e.g., brain tumors (glioma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma), solid tumors (nasopharyngeal cancer, basal cellcarcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer,primary liver cancer or endometrial cancer, and tumors of the vascularsystem (angiosarcoma and hemangiopericytoma), osteoporosis, hepatitis,HIV, AIDS, spondylarthritis, rheumatoid arthritis, inflammatory boweldiseases (IBD), sepsis and septic shock, Crohn's Disease, psoriasis,schleraderma, graft versus host disease (GVHD), allogenic islet graftrejection, hematologic malignancies, such as multiple myeloma (MM),myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML),inflammation associated with tumors, peripheral nerve injury ordemyelinating diseases. In certain embodiments, the use of themedicament is for the treatment of plaque psoriasis, ulcerative colitis,non-Hodgkin's lymphoma, breast cancer, colorectal cancer, juvenileidiopathic arthritis, macular degeneration, respiratory syncytial virus,Crohn's disease, rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, osteoporosis, treatment-induced bone loss, bone metastases,multiple myeloma, Alzheimer's disease, glaucoma, and multiple sclerosis.In certain embodiments that may be combined with any of the precedingembodiments, the use of the medicament further comprises apharmaceutically acceptable excipient. In certain embodiments that maybe combined with any of the preceding embodiments, the immunoglobulinconjugate in the medicament shows at least at least eighty percent, atleast eighty-five percent, at least ninety percent, at least ninety-fivepercent, at least ninety-six percent, at least ninety-seven percent, atleast ninety-eight percent, or at least ninety-nine percentnon-oligomerized monomer. In certain embodiments, the oligomerization ismeasured by non-reducing SDS-PAGE.

EXAMPLES

The Examples described herein refer to particular, non-limitingembodiments of the invention.

Example 1 Design, Expression, and Conjugation of Antibody CysteineVariants

A set of IgG1 cysteine variants was designed such that eachimmunoglobulin fold domain is represented (Table 1). Variants 1-13 weredesigned from the X-ray structure of antibody-1. Variant 14 was selectedfrom the structure of another IgG1, antibody-2, built by homologymodeling with respect to antibody-1. All sites were exposed on theantibody surface. Polar residues, such as serine and threonine andarginine, or charged residues, such as lysine, were substituted withcysteine. The light and heavy chain genes were subcloned in vector gWIZ(Genlantis) and engineered for protein expression by transienttransfection of mammalian cells. Antibody variants were either de novosynthesized (GeneArt) or generated by site-directed mutagenic PCR andconfirmed by sequencing. Antibody wild type and variants were expressedat 10-100 mg levels by transient transfection of Freestyle HEK 293 cells(Invitrogen) with polyethyleneimine (Polysciences) as the transfectionreagent. Cell culture supernatant was collected 7-10 dayspost-transfection. Antibodies were purified on a protein A column (GEHealthcare), eluted with 50 mM citrate buffer, pH 3.5, and bufferexchange in 100 mM Tris pH 7.0 buffer for fluorescence labeling.

Following expression and purification of antibody variants, theengineered surface cysteines were mostly oxidized. For example bothVariant 4 and 6 had less than 0.3 free thiol per antibody molecule asopposed to the anticipated 2.0 for the antibodies with engineeredsurface cysteines. We compared the effect of a mild reducing agent, TCEP(Tris[2-carboxyethyl]phosphine hydrochloride) and a stronger reducingagent, DTT (dithiothreitol) on a variant from class I and a variant fromclass IV. Initially, the non-oligomerizing Variant 4 showed 0.13 freethiol per antibody, and the highly oligomerizing Variant 6 had 0.25 freethiol per antibody. Aliquots of wild type, variant 4, and variant 6 weretreated in five different conditions: 1) no reducing agent, 2) TCEP,10×, 1 hour, 3) TCEP, 20×, 1 hour, 4) DTT, 5×, 15 minutes, and 5) DTT,10×, 15 minutes. After removal of the reducing agent the samples wereresolved on non-reducing PAGE and were quantified for free thiol. Acomparison of the results for wild type and variants indicated that theTCEP treatment was sufficient to reduce cysteines in non-oligomerizedform (Variant 4) with little effect on WT. However, cysteines fromoligomers (Variant 6) were reduced only after a harsher treatment.Treatment with DTT even at low levels leads to antibody fragmentationfor WT and both variants. The sites where the surface cysteines wereintroduced had a profound effect on the ability to decap the engineeredcysteines for conjugation.

Different methods were attempted for the specific reduction of theengineered surface thiols before labeling. TCEP and DTT were two of thereagent used, and levels of free thiol were quantified using Ellman'sreagent (Invitrogen). We found L-cysteine to work best in oursite-specific labeling experiments, so the following two-step protocolwas used. First, the variants were incubated with 100-200 fold excess ofL-cysteine for 4 hrs at 37° C., followed by buffer exchange into 50 mMTris/EDTA. Second, the samples were incubated with 5-10 fold excess ofAlexa488 maleimide dye (Invitrogen) for 1 hr at room temperature or with10 fold excess of Pyrene maleimide dye (Invitrogen) for 12 hrs at roomtemperature. After removal of free dye, and buffer exchange to 50 mMphosphate buffer pH 7.0, the efficiency of protein labeling wascalculated as mole of dye per mole of protein according tomanufacturer's protocols (Invitrogen).

Example 2 Characterization of the Engineered Antibody Cysteine Variants

Unlabeled and labeled antibody samples were analyzed by SDS-PAGE. Gelsof 7.5%, 10%, and 12% were used for non-reducing analysis. Gels of 12%were used for reducing analysis of heated samples with DTT. Usually,samples of 5-10 μg were loaded per lane. Fluorescent images were takenunder UV light before staining with Coomassie Blue. Antibody digestionwas carried out by GluC (1:20 wt enzyme per wt antibody, at 25° C. for12-24 hrs) and pronase (1:20 wt enzyme per wt antibody, at 37° C. for 1hr).

Non-reducing gels show monomers as well as the presence of dimers,trimers, and in some cases even higher oligomers. Reducing gels show theexclusive labeling of the light or heavy chain depending on where thesurface cysteine was engineered. Labeled and unlabeled variants 1-6 werealso analyzed for antigen binding specificity. The variants retainactivity within 80% and 130% of wild type with some loss of activityupon labeling. Unlabeled variant 1 retained approximately 110% ofwild-type activity, whereas labeled variant 1 retained approximately80%. Unlabeled variant 2 retained approximately 105% activity ofwild-type, whereas labeled variant 2 retained slightly less than 100%activity. Unlabeled and labeled variant 3 both retained approximately110% of wild-type activity. Unlabeled variant 4 retained approximately125% of wild-type activity, whereas labeled variant 4 retainedapproximately 95% of activity. Unlabeled variant 5 retainedapproximately 120% of wild-type activity, whereas labeled variant 6retained approximately 100% of activity. Finally, unlabeled variant 6retained approximately 115% of wild-type activity, whereas labeledvariant 6 retained approximately 90% of activity. Similarly to itsunlabeled counterpart, labeled variant 6 showed high oligomerizationpropensity.

Most variants were labeled near the optimal efficiency of 2.0 moles dyeper mole antibody (two identical cysteines per antibody molecule).Higher than 2.0 labeling efficiency is non-desirable since that wouldsuggest partial disruptions and labeling of intrachain disulfides.Variants with high oligomerization propensity such as Variant 6, Variant11 and Variant 5 did not label as efficiently. Even among the othervariants, labeling conditions such as time of reaction and dye toprotein ratio had to be optimized on an individual basis because not allengineered cysteines were equally amenable to conjugation. Variants 1-14were specifically labeled at the chain that carries the engineeredcysteine. Proteolytic treatment of the variants with pronase yieldeddifferent fluorescence patterns for most variants, but similar patternsfor variants with neighboring substitutions, such as Variant 3 and 12.Thus, most variants were efficiently and specifically labeled.

Five classes of cross-linking propensity were distinguished for this setof cysteine variants (Table 1). Class I comprises variants that weremonomeric and remain stable after labeling. Variants of class IIcontained a small percent of dimers before and after labeling. Class IIIvariants had a more pronounced propensity to oligomerize includingformation of some trimers. Class IV variants had an even higherpropensity to oligomerize as evidenced by the presence of aggregateslarger than trimer, especially after labeling. Class V included variantsof high oligomerization propensity similarly to variant of Class IV withadditional structural abnormalities such as fragmentation or colorationof purified concentrated sample.

Example 3 Application of the Engineered Antibody Cysteine Variants

Cysteine variants with low cross-linking propensity (Variants 1-4, 7,10, 12-14) were labeled with high specificity and efficiency and littleoligomerization. Labeling with maleimide dyes is only one example ofsite-specific conjugation on these antibody variants. Molecules withmany other functionalities such as binding specificity or toxicity canbe equally attached. Thus, this set of variants expands on therepertoire of antibody variants to serve for payload vehicles intargeted therapy or for in vitro and in vivo fluorescence analysis.

To illustrate the fluorescence application of one of the variants, weanalyzed the emission pattern of variants conjugated with thefluorophore pyrene. When two pyrene molecules are close together thereis a characteristic increase of emission at 465 nm known as excimerfluorescence. We labeled Variants 4 and 7 with pyrene maleimide andmonitored emission spectra. While Variant 4 showed basal level emissionat 465 nm, Variant 7 showed strong excimer fluorescence. Considering theposition of the engineered cysteine in C_(H)1 for Variant 7, on theinner side of the Fab domains, the observed result correlates with theknown scissoring effect of the Fab's with respect to Fc. Thus, thisvariant can be used in the analysis of antibody domain dynamics.

The high oligomerization propensity of Variant 6 suggested anotherutility of antibody cysteine variants that was explored in greaterdetail. Labeled variant 6 was subjected to gel filtration chromatographyin order to separate monomer from oligomers, and protein-containingfractions were resolved on a 7.5% non-reducing SDS-PAGE gel and analyzedbefore and after staining with Coomasssie Blue. The gel filtrationanalysis on variant 6 indicated a competition between labeling andcrosslinking: the higher the MW of the species, the lower the labelingefficiency (indicated by the level of fluorescence). The highest MWspecies had a labeling efficiency of 0.5, while the monomeric specieshad a labeling efficiency of 1.0, with the original labeled sample oflabeling efficiency 0.8. An antibody variant with multiple oligomers,Variant 6 presents an excellent control for antibody oligomerization anda suitable standard for high molecular weight proteins, with theadditional functionality that it can be site-specifically labeled.

Example 4 Correlation between Cross-Linking Propensity (CLP) andSpatial-Aggregation Propensity (SAP) of the Cysteine Variants

Cross-linking propensity (CLP) and spatial aggregation propensity (SAP)were compared for the cysteine variants where specific amino acids aresubstituted with cysteine. Each variant was assigned CLP based onnon-reducing SDS-PAGE analysis. SAP values for the mutated residues arefrom computational results with radius of 5 Å. We overlaid theengineered cysteine variants on the SAP-coded antibody-1 structure.

The following correlations were observed. All amino acids substitutedwith cysteines are of negative SAP-value in the range from −0.27 to0.00. This is consistent with the choice of polar or charged amino acidsfor substitution. All variants of CLP class I have SAP between 0.00 and−0.11 (Variants 3, 4, 7, 10, 12), and all variants of CLP class II haveSAP between −0.12 and −0.23 (Variants 1, 2, 13). However, there arevariants with SAP in those ranges with high CLP (Variants 8, 9 and 11for example). The highly cross-linking variants Variant 8 and 11neighbor high-SAP sites. Variant 5 with CLP III is adjacent to high-SAPsites in C_(H)2, while Variant 2 of CLP II is not. However, there is nosuch correlation between Variant 6 and Variant 10 in C_(H)3, and betweenVariant 9 and 14 in V_(H).

An additional observation was made of Variant 14. Variant 14 fails toexpress if there is a region of high SAP nearby, whereas it expresseswhen this high SAP region is replaced by a region of low SAP. A 100-foldhigher yield of Variant 14 in the stabilized antibody-2 (35.6 mg/Lculture) was observed compared to that of Variant 14 in the nativeantibody-2 background (0.34 mg/L). The relative yield of Variant 14 inthe different backgrounds indicated a structural problem when a cysteineis introduced on the surface of a protein near a region of high SAPvalue. The problem was resolved when two hydrophobic amino acids in thehydrophobic patch neighboring the engineered cysteine were substitutedwith lysines.

In summary, correlations exist between stability of cysteine variantsand SAP: 1) cysteine variants with low cross-linking propensity haveslightly negative SAP (0.00 to −0.11), 2) cysteine variants with morenegative SAP (−0.12 to −0.23) are more prone to cross-linking, and 3)cysteine variants in immediate proximity to patches of high-SAP are morelikely to cross-link or have structural abnormalities. Conclusions 1 and2 are consistent with the previously defined notion that fully exposedresidues may be more susceptible to cross-linking [9].

Example 5 Conclusion

We designed a set of human IgG1 cysteine variants that are broadlydistributed on the antibody molecule with at least one variant perimmunoglobulin fold domain. Most of these variants are stable, and canbe conjugated efficiently and specifically without significant loss ofantigen binding activity. Thus, the stable antibody variants add to therepertoire of variants for site-specific conjugation of payloadmolecules. If fluorophores are attached to the engineered cysteines, thedynamics of particular domains can be analyzed. The highly oligomerizingvariants are beneficial as well, as the numerous multimers provide aconvenient standard for antibody aggregates and for high molecularweight proteins in general.

A correlation between the cross-linking propensity of the antibodycysteine variants described here and the SAP method demonstrate that theSAP methodology may be used to screen for conjugation sites with reducedcross-linking. The SAP technology is computer-based, so it reduces thetime and experimental work in variants design. CLP/SAP comparison showedthat substitution of partially and not fully exposed amino acids yieldsthe most stable variants. Moreover, the comparison showed thatneighboring hydrophobic patches should be avoided.

The engineered human IgG1 surface cysteine variants described hereprovide new sites for site-specific conjugation of therapeuticantibodies and methods for identifying further variants. The variantswith little crosslinking propensity have the greatest utility indeveloping antibodies for targeted therapy. The cysteine variantsdisclosed herein include new sites in previously represented domains(C_(L), C_(H)1, C_(H)3) as well as in previously unrepresented domains(V_(L), V_(H), C_(H)2).

Moreover, the labeled variants can be used as a set of site-specificfluorescent antibody markers for in vitro and in vivo laboratoryresearch. The fluorescently labeled products can be commercialized viabiotechnology companies (such as Thermo Scientific Pierce, GEHealthcare, and Invitrogen) providing the research community withantibody and other protein reagents.

The highly cross-linking variant 6 is a useful protein-gel or otherchromatography technique standard. It can be marketed by companies (forexample Invitrogen, Bio-Rad, and Pierce) providing protein reagents.

The correlation between CLP and SAP further suggested a commercialapplication of our previously described SAP technology. Consideration ofSAP can improve the design of stable antibody cysteine variants forsite-specific conjugation.

TABLE 1 Variant Domain Residue CLP SAP 1 CH2 K248 II −0.12 2 CH2 K326 II−0.19 3 VL T22 I −0.07 4 CL T197 I −0.03 5 CH2 N286 III −0.27 6 CH3 S440IV −0.09 7 CH1 S125 I −0.06 8 CH2 S298 V −0.19 9 VH S25 III −0.07 10 CH3S442 I 0.00 11 CH2 S254 V −0.06 12 VL T20 I −0.04 13 CH3 S415 II −0.2314 VH S7 I −0.11

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1. (canceled)
 2. An immunoglobulin conjugate, comprising animmunoglobulin having at least one mutation at a residue selected fromthe group consisting of 20(V_(L)), 22(V_(L)), 25(V_(H)), 125(C_(H1)),248(C_(H2)), 254(C_(H2)), 286(C_(H2)), 298(C_(H2)), and 326(C_(H2)),wherein the at least one mutation is a substitution with a cysteineresidue, and an atom or molecule, wherein the atom or molecule isconjugated to the cysteine residue.
 3. The immunoglobulin conjugate ofclaim 2, further comprising a linker molecule having at least tworeactive sites, wherein a first reactive site is bound to the cysteineresidue of the immunoglobulin and a second reactive site is bound to theatom or molecule.
 4. The immunoglobulin conjugate of claim 3, whereinthe linker molecule is selected from the group consisting of ahydrazone, a disulfide, a peptide, a chelating agent, and a maleimide.5. The immunoglobulin conjugate of claim 3, wherein the linker moleculeforms a disulfide linkage with the cysteine residue.
 6. Theimmunoglobulin conjugate of claim 2, wherein the atom or molecule isselected from the group consisting of a radionuclide, a chemotherapeuticagent, a microbial toxin, a plant toxin, a polymer, a carbohydrate, acytokine, a fluorescent label, a luminescent label, an enzyme-substratelabel, an enzyme, a peptide, a peptidomimetic, a nucleotide, an siRNA, amicroRNA, an RNA mimetic, and an aptamer.
 7. The immunoglobulinconjugate of claim 2, wherein the atom or molecule is selected from thegroup consisting of ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹⁷⁷Lu, ²¹³Bi, ²¹¹At, acalicheamicin, a duocarmycin, a maytanisoid, an auristatin, ananthracyclin, Pseudomonas exotoxin A, Diptheria toxin, ricin,polyethylene glycol, hydroxyethyl starch, and a mannosyl residue.
 8. Theimmunoglobulin conjugate of claim 2, wherein the immunoglobulinconjugate further comprises an antigen binding activity and the activityis at least eighty percent, at least ninety percent, at least onehundred percent, at least one hundred ten percent, at least one hundredtwenty percent, or at least one hundred thirty percent of the antigenbinding activity of the unmutated immunoglobulin.
 9. A modified orisolated immunoglobulin comprising at least one mutation at a residueselected from the group consisting of 20(V_(L)), 22(V_(L)), 25(V_(H)),125(C_(H1)), 248(C_(H2)), 254(C_(H2)), 286(C_(H2)), and 326(CH₂),wherein the at least one mutation is a substitution with a cysteineresidue.
 10. An isolated or recombinant polynucleotide encoding theimmunoglobulin of claim
 9. 11. A vector comprising the polynucleotide ofclaim 10 operably linked to an inducible promoter.
 12. A host cellcomprising the vector of claim
 11. 13. A method of producing animmunoglobulin, comprising: (a) providing a culture medium comprisingthe host cell of claim 12; and (b) placing the culture medium inconditions under which the immunoglobulin is expressed.
 14. A method ofproducing an immunoglobulin conjugate, comprising: (a) providing theimmunoglobulin of claim 9; (b) reducing the one or more substitutedcysteine residues with a reducing agent to form reduced cysteineresidues; and (c) incubating the immunoglobulin with an atom ormolecule, wherein the atom or molecule is reactive with the reducedcysteine residues, to form an immunoglobulin conjugate.
 15. A method forreducing the cross-linking between surface-exposed cysteines of animmunoglobulin in a highly concentrated pharmaceutical formulation ofimmunoglobulin conjugates, comprising: (a) providing an immunoglobulin;(b) substituting a residue selected from the group consisting of7(V_(H)), 20(V_(L)), 22(V_(L)), and 125(C_(H1)) with a cysteine residue,(c) reducing the one or more substituted cysteine residues with areducing agent to form reduced cysteine residues; (d) incubating theimmunoglobulin with an atom or molecule, wherein the molecule isreactive with the reduced cysteine residues, to form an immunoglobulinconjugate; and (e) generating a highly concentrated, liquid formulationof the immunoglobulin conjugate wherein the immunoglobulin conjugateconcentration is at least 20 mg/ml, at least 30 mg/ml, at least 40mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml, atleast 125 mg/ml, or at least 150 mg/ml.
 16. The method of claim 15,wherein the immunoglobulin conjugate comprises an antigen bindingactivity and the activity is at least eighty percent, at least ninetypercent, at least one hundred percent, at least one hundred ten percent,at least one hundred twenty percent, or at least one hundred thirtypercent of the antigen binding activity of the unmutated immunoglobulin.17. A pharmaceutical composition comprising the immunoglobulin conjugateof claim 2 and a pharmaceutically acceptable excipient, wherein at leasteighty percent, at least eighty-five percent, at least ninety percent,at least ninety-five percent, at least ninety-six percent, at leastninety-seven percent, at least ninety-eight percent, or at leastninety-nine percent of the immunoglobulin conjugate is non-oligomerizedmonomer.
 18. The pharmaceutical composition of claim 17, wherein theimmunoglobulin conjugate is at a concentration of at least 10 mg/ml, atleast 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml,at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least150 mg/ml.
 19. A method for selecting a residue of an immunoglobulin formutation to cysteine, comprising: (a) calculating theSpatial-Aggregation-Propensity of a first amino acid residue on thesurface of the immunoglobulin; (b) calculating theSpatial-Aggregation-Propensities of a plurality of residues of theimmunoglobulin within immediate proximity of the first residue; and (c)selecting the first amino acid residue for mutation to cysteine if theSpatial-Aggregation-Propensity of the first amino acid residue is equalto or in between the values of 0 and −0.11 and if the plurality ofresidues has Spatial-Aggregation-Propensities of less than
 0. 20. Amodified or isolated immunoglobulin comprising at least one mutation ofa surface-exposed residue to cysteine, wherein theSpatial-Aggregation-Propensity of the residue is equal to or in betweenthe values of 0 and −0.11 and wherein theSpatial-Aggregation-Propensities of a plurality of residues of theimmunoglobulin within immediate proximity of the first residue haveSpatial-Aggregation-Propensities of less than 0.